Fused ring heterocycle kinase modulators

ABSTRACT

The present invention provides novel fused ring heterocycle kinase modulators and methods of using the novel fused ring heterocycle kinase modulators to treat diseases mediated by kinase activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 11/192,318, filed Jul. 27, 2005, now U.S. Pat. No.7,452,993 and to which application we claim priority under 35 U.S.C.§121, and claims the benefit of U.S. Provisional Patent Application No.60/591,778, filed Jul. 27, 2004, U.S. Provisional Patent Application No.60/680,091, filed May 11, 2005, and U.S. Provisional Patent ApplicationNo. 60/591,886, filed Jul. 27, 2004. The disclosures of each of theseapplications are incorporated herein by reference in their entirety forall purposes.

BACKGROUND OF THE INVENTION

Mammalian protein kinases are important regulators of cellularfunctions. Because dysfunctions in protein kinase activity have beenassociated with several diseases and disorders, protein kinases aretargets for drug development.

The tyrosine kinase receptor, FMS-like tyrosine kinase 3 (FLT3), isimplicated in cancers, including leukemia, such as acute myeloidleukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplasia.About one-quarter to one-third of AML patients have FLT3 mutations thatlead to constitutive activation of the kinase and downstream signalingpathways. Although in normal humans, FLT3 is expressed mainly by normalmyeloid and lymphoid progenitor cells, FLT3 is expressed in the leukemiccells of 70-80% of patients with AML and ALL. Inhibitors that targetFLT3 have been reported to be toxic to leukemic cells expressing mutatedand/or constitutively-active FLT3. Thus, there is a need to developpotent FLT3 inhibitors that may be used to treat diseases and disorderssuch as leukemia.

The Abelson non-receptor tyrosine kinase (c-Abl) is involved in signaltransduction, via phosphorylation of its substrate proteins. In thecell, c-Abl shuttles between the cytoplasm and nucleus, and its activityis normally tightly regulated through a number of diverse mechanisms.Abl has been implicated in the control of growth-factor and integrinsignaling, cell cycle, cell differentiation and neurogenesis, apoptosis,cell adhesion, cytoskeletal structure, and response to DNA damage andoxidative stress.

The c-Abl protein contains approximately 1150 amino-acid residues,organized into a N-terminal cap region, an SH3 and an SH2 domain, atyrosine kinase domain, a nuclear localization sequence, a DNA-bindingdomain, and an actin-binding domain.

Chronic myelogenous leukemia (CML) is associated with the Philadelphiachromosomal translocation, between chromosomes 9 and 22. Thistranslocation generates an aberrant fusion between the bcr gene and thegene encoding c-Abl. The resultant Bcr-Abl fusion protein hasconstitutively active tyrosine-kinase activity. The elevated kinaseactivity is reported to be the primary causative factor of CML, and isresponsible for cellular transformation, loss of growth-factordependence, and cell proliferation.

The 2-phenylaminopyrimidine compound imatinib (also referred to asSTI-571, CGP 57148, or Gleevec) has been identified as a specific andpotent inhibitor of Bcr-Abl, as well as two other tyrosine kinases,c-kit and platelet-derived growth factor receptor. Imatinib blocks thetyrosine-kinase activity of these proteins. Imatinib has been reportedto be an effective therapeutic agent for the treatment of all stages ofCML. However, the majority of patients with advanced-stage or blastcrisis CML suffer a relapse despite continued imatinib therapy, due tothe development of resistance to the drug. Frequently, the molecularbasis for this resistance is the emergence of imatinib-resistantvariants of the kinase domain of Bcr-Abl. The most commonly observedunderlying amino-acid substitutions include Glu255Lys, Thr315Ile,Tyr293Phe, and Met351Thr.

MET was first identified as a transforming DNA rearrangement (TPR-MET)in a human osteosarcoma cell line that had been treated withN-methyl-N′-nitro-nitrosoguanidine (Cooper et al. 1984). The METreceptor tyrosine kinase (also known as hepatocyte growth factorreceptor, HGFR, MET or c-Met) and its ligand hepatocyte growth factor(“HGF”) have numerous biological activities including the stimulation ofproliferation, survival, differentiation and morphogenesis, branchingtubulogenesis, cell motility and invasive growth. Pathologically, METhas been implicated in the growth, invasion and metastasis of manydifferent forms of cancer including kidney cancer, lung cancer, ovariancancer, liver cancer and breast cancer. Somatic, activating mutations inMET have been found in human carcinoma metastases and in sporadiccancers such as papillary renal cell carcinoma. The evidence is growingthat MET is one of the long-sought oncogenes controlling progression tometastasis and therefore a very interesting target. In addition tocancer there is evidence that MET inhibition may have value in thetreatment of various indications including: Listeria invasion,Osteolysis associated with multiple myeloma, Malaria infection, diabeticretinopathies, psoriasis, and arthritis.

The tyrosine kinase RON is the receptor for the macrophage stimulatingprotein and belongs to the MET family of receptor tyrosine kinases. LikeMET, RON is implicated in growth, invasion and metastasis of severaldifferent forms of cancer including gastric cancer and bladder cancer.

The Aurora family of serine/theronine kinases is essential for mitoticprogression. Expression and activity of the Aurora kinases are tightlyregulated during the cell cycle. A variety of proteins having roles incell division have been identified as Aurora kinase substrates. Based onthe known function of the Aurora kinases, inhibition of their activityis believed to disrupt the cell cycle and block proliferation andtherefore tumor cell viability. Harrington et al., Nature Medicine,advanced publication online (2004).

3-Phosphoinositide-dependent kinase 1 (PDK1) is a Ser/Thr protein kinasethat can phosphorylate and activate a number of kinases in the AGCkinase super family, including Akt/PKB, protein kinase C (PKC),PKC-related kinases (PRK1 and PRK2), p70 ribobsomal S6-kinase (S6K1),and serum and glucocorticoid-regulated kinase (SGK). The firstidentified PDK1 substrate is the proto-oncogene Akt. Numerous studieshave found a high level of activated Akt in a large percentage (30-60%)of common tumor types, including melanoma and breast, lung, gastric,prostate, hematological and ovarian cancers. The PDK1/Akt signalingpathway thus represents an attractive target for the development ofsmall molecule inhibitors that may be useful in the treatment of cancer.Feldman et al., JBC Papers in Press. Published on Mar. 16, 2005 asManuscript M501367200.

Because kinases have been implicated in numerous diseases andconditions, such as cancer, there is a need to develop new and potentprotein kinase inhibitors that can be used for treatment. The presentinvention fulfills these and other needs in the art. Although certainprotein kinases are specifically named herein, the present invention isnot limited to inhibitors of these kinases, and, includes, within itsscope, inhibitors of related protein kinases, and inhibitors ofhomologous proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the wild-type ABL numbering according to ABL exon Ia.

BRIEF SUMMARY OF THE INVENTION

It has been discovered that, surprisingly, fused ring heterocyclecompounds of the present invention may be used to modulate kinaseactivity and to treat diseases mediated by kinase activity. These novelfused ring heterocycle kinase modulators are described in detail below.In addition, inhibitory activities of selected compounds are disclosedherein.

In one aspect, the present invention provides a fused ring heterocyclekinase modulator having the formula:

In Formula (I), L¹ and L² are independently a bond, —S(O)_(n)—, —O—,—NH—, unsubstituted C₁-C₅ alkylene, or unsubstituted 2 to 5 memberedheteroalkylene. The symbol n is an integer from 0 to 2. R¹ and R² areindependently substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl,or substituted or unsubstituted aryl. In some embodiments, R¹ is notsubstituted or unsubstituted pyrrolyl. In other embodiments, L¹ is notunsubstituted 2 to 5 membered heteroalkylene when R¹ and R² are bothunsubstituted phenyl. In other embodiments, L¹ is not —S(O)₂— where R²is unsubstituted piperazinyl.

In another aspect, the present invention provides a fused ringheterocycle kinase modulator (also referred to herein as a “compound ofthe present invention”) having the formula:

In Formula (II), L¹, L², R¹, and R² are as defined above in thediscussion of Formula (I).

In another aspect, the present invention provides a fused ringheterocycle kinase modulator (also referred to herein as a “compound ofthe present invention”) having the formula:

In Formula (III), L¹, L², R¹, and R² are as defined above in thediscussion of Formula (I).

In another aspect, the present invention provides methods of modulatingprotein kinase activity using the fused ring heterocycle kinasemodulators of the present invention. The method includes contacting theprotein kinase with a fused ring heterocycle kinase modulator.

In another aspect, the present invention provides methods of treating adisease mediated by kinase activity (kinase-mediated disease ordisorder) in a subject (e.g. mammals, such as humans) in need of suchtreatment. The method includes administering to the subject atherapeutically effective amount of a fused ring heterocycle kinasemodulator of the present invention.

In another aspect, the present invention provides a pharmaceuticalcomposition including a fused ring heterocycle kinase modulator inadmixture with a pharmaceutically acceptable excipient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—, —CH₂C≡CCH₂—, and—CH₂CH₂CH(CH₂CH₂CH₃)CH₂—. Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen, sulfur,and phosphorus atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxo,alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)OR′—represents both —C(O)OR′— and —R′OC(O)—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′ R′ or the like, it will be understood that the terms heteroalkyland —NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms (in each separate ring in the caseof multiple rings) selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a carbon or heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent radicals of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like). However, theterm “haloaryl,” as used herein is meant to cover only aryls substitutedwith one or more halogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” referrersto a carbon or heteroatom.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” and“heterocycloalkyl”, “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)OR′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxo, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on aromatic ring system; andwhere R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B-, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. The amino group is —NR′R″, wherein R′ and R″ aretypically selected from hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The terms “a,” “an,” or “a(n)”, when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

Description of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The terms “treating” or “treatment” in reference to a particular diseaseincludes prevention of the disease.

The symbol

denotes the point of attachment of a moiety to the remainder of themolecule.

I. Fused Ring Heterocycle Kinase Modulators

In one aspect, the present invention provides a fused ring heterocyclekinase modulator (also referred to herein as a “compound of the presentinvention”) having the formula:

In Formula (I), L¹ and L² are independently a bond, —S(O)_(n)—, —O—,—NH—, substituted or unsubstituted C₁-C₅ alkylene, or substituted orunsubstituted 2 to 5 membered heteroalkylene. In some embodiments, L¹and L² are independently a bond, —S(O)_(n)—, —O—, —NH—, unsubstitutedC₁-C₅ alkylene, or unsubstituted 2 to 5 membered heteroalkylene. Thesymbol n is an integer from 0 to 2. R¹ and R² are independentlysubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted aryl.

In some embodiments, R¹ is not substituted or unsubstituted pyrrolyl. Inother embodiments, L¹ is not unsubstituted 2 to 5 memberedheteroalkylene when R¹ and R² are both unsubstituted phenyl. In otherembodiments, L¹ is not —S(O)₂— where R² is unsubstituted piperazinyl.

In some embodiments, R¹ is not substituted or unsubstituted 5-memberedheteroaryl. In other embodiments R¹ is substituted or unsubstituted6-membered heteroaryl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R¹ may also be a substituted or unsubstituted 6-memberedheteroaryl, or substituted or unsubstituted aryl.

In other embodiments, L¹ is not unsubstituted 2 to 5 memberedheteroalkylene when R¹ and R² are both unsubstituted aryl. In otherembodiments, L¹ is not unsubstituted 2 to 5 membered heteroalkylene whenR¹ and R² are both substituted or unsubstituted phenyl. In otherembodiments, L¹ is selected from a bond, —S(O)_(n)—, —O—, —NH—, andunsubstituted C₁-C₅ alkylene. In other embodiments, n is 0 or 1. Inother embodiments, L¹ is selected from a bond, —O—, —NH—, andunsubstituted C₁-C₅ alkylene.

In some embodiments, L¹ is not —S(O)₂— where R² is substituted orunsubstituted piperazinyl. In other embodiments, L¹ is not —S(O)₂— whereR² is unsubstituted heterocycloalkyl. In other embodiments, L¹ is not—S(O)₂— where R¹ is substituted or unsubstituted heterocycloalkyl. Inother embodiments, n is 0 or 1. In other embodiments, L¹ is not —S(O)₂—where L² is a bond.

In some embodiments, R² is not an unsubstituted 6-memberedheterocycloalkyl. In other embodiments, R² is not a substituted orunsubstituted 6-membered heterocycloalkyl. In other embodiments, R² isselected from substituted or unsubstituted heteroaryl, substituted orunsubstituted 5-membered heterocycloalkyl, substituted or unsubstitutedaryl, and substituted or unsubstituted cycloalkyl. R² may also besubstituted or unsubstituted cycloalkyl, substituted heterocycloalkyl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedaryl.

In some embodiments, R¹ is not substituted or unsubstituted isoxazolylwhere R² is unsubstituted pyridinyl. In other embodiments, R¹ is notsubstituted or unsubstituted isoxazolyl where L¹ is a bond or —CH₂—. Inother embodiments, R¹ is not substituted or unsubstituted isoxazolyl. Inother embodiments, R¹ is not a 4-substituted isoxazolyl. In otherembodiments, R¹ is not a 5-yl-isoxazolyl. In other embodiments, R¹ isnot a 4-substituted-5-yl-isoxazolyl. In other embodiments, R¹ is not anisoxazolyl substituted with a fluoro-substituted aryl.

L¹ and L² may independently be a bond, —S(O)_(n)—, —O—, —NH—, orunsubstituted C₁-C₅ alkylene. In some embodiments, L¹ and L² are a bond.In other embodiments, L¹ or L² is a bond.

R¹ may be a substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted 5- or6-membered heteroaryl, or substituted or unsubstituted aryl. R¹ may alsobe a substituted or unsubstituted 6-membered heteroaryl, or substitutedor unsubstituted aryl.

In other embodiments, R¹ is (1) unsubstituted C₃-C₇ cycloalkyl; (2)unsubstituted 3 to 7 membered heterocycloalkyl; (3) unsubstitutedheteroaryl; (4) unsubstituted aryl; (5) substituted C₃-C₇ cycloalkyl;(6) substituted 3 to 7 membered heterocycloalkyl; (7) substituted aryl;or (8) substituted heteroaryl. In some related embodiments, (5) and (6)are substituted with an oxo, —OH, —CF₃, —COOH, cyano, halogen,R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl,R¹²-substituted or unsubstituted heteroaryl, -L¹²-C(X¹)R⁷, -L¹²-OR⁸,-L¹²NR⁹¹R⁹², or -L¹²-S(O)_(m)R¹⁰. X¹ is ═S, ═O, or ═NR¹⁵, wherein R¹⁵ isH, —OR¹⁵¹, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, or R¹²-substituted or unsubstituted heteroaryl. R¹⁵¹is hydrogen or R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl. The symbolm is an integer from 0 to 2.

In other related embodiments, (7) and (8) are substituted with an —OH,—CF₃, —COOH, cyano, halogen, R¹¹-substituted or unsubstituted C₁-C₁₀alkyl, R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl,-L¹²-C(X¹)R⁷, -L²-OR⁸, -L¹²-NR⁹¹R⁹², or -L¹²-S(O)_(m)R¹⁰. L¹² is a bond,unsubstituted C₁-C₁₀ alkylene, or unsubstituted heteroalkylene. X¹ and mare as defined above.

R⁷ is hydrogen, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl, —OR⁷¹,or —NR⁷²R⁷³. R⁷¹, R⁷², and R⁷³ are independently hydrogen,R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl, orR¹²-substituted or unsubstituted heteroaryl. R⁷² and R⁷³ are optionallyjoined with the nitrogen to which they are attached to form anR¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR¹²-substituted or unsubstituted heteroaryl.

R⁸, R⁹¹ and R⁹² are independently hydrogen, —CF₃, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, R¹²-substituted or unsubstitutedheteroaryl, —C(X²)R⁸¹, or —S(O)_(w)R⁸¹. X² is ═S, ═O, or ═NR¹⁶. R¹⁶ isR¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl, orR¹²-substituted or unsubstituted heteroaryl. The symbol w is an integerfrom 0 to 2. R⁹¹ and R⁹² are optionally joined with the nitrogen towhich they are attached to form an R¹¹-substituted or unsubstituted 3 to7 membered heterocycloalkyl, or R¹²-substituted or unsubstitutedheteroaryl.

R⁸¹ is hydrogen, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl, or—NR⁸¹¹R⁸¹².

R⁸¹¹ and R⁸¹² are independently hydrogen, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, or R¹²-substituted orunsubstituted heteroaryl. R⁸¹¹ and R⁸¹² are optionally joined with thenitrogen to which they are attached to form an R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R¹²-substituted orunsubstituted heteroaryl.

In some embodiments, R⁸¹ and R¹⁶ are optionally joined with the atoms towhich they are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁸¹¹ and R¹⁶ are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁸¹ and R⁹² are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁸¹¹ and R⁹² are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

R¹⁰ is hydrogen, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl, or—NR¹⁰¹R¹⁰². R¹⁰¹ and R¹⁰² are independently hydrogen, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, or R¹²-substituted orunsubstituted heteroaryl. R¹⁰¹ and R¹⁰² are optionally joined with thenitrogen to which they are attached to form an R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R¹²-substituted orunsubstituted heteroaryl.

R¹¹ is oxo, —OH, —COOH, —CF₃, —OCF₃, —CN, amino, halogen,R¹³-substituted or unsubstituted 2 to 10 membered alkyl, R¹³-substitutedor unsubstituted 2 to 10 membered heteroalkyl, R¹³-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹³-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹⁴-substituted or unsubstituted aryl, orR¹⁴-substituted or unsubstituted heteroaryl.

R¹² is —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, R¹³-substituted orunsubstituted 2 to 10 membered alkyl, R¹³-substituted or unsubstituted 2to 10 membered heteroalkyl, R¹³-substituted or unsubstituted C₃-C₇cycloalkyl, R¹³-substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, R¹⁴-substituted or unsubstituted aryl, orR¹⁴-substituted or unsubstituted heteroaryl.

R¹³ is oxo, —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.

R¹⁴ is —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.

In some embodiments, R¹ is (1), (2), (4), (5), (6), or (7) (i.e.unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7 memberedheterocycloalkyl, unsubstituted aryl, substituted C₃-C₇ cycloalkyl,substituted 3 to 7 membered heterocycloalkyl, or substituted aryl,respectively). In some embodiments, where R¹ is (3), or (8), then theheteroaryl is a 6-membered heteroaryl.

Where R¹ is (7) or (8) (i.e. substituted aryl or substitutedheteroaryl), (7) and (8) may be substituted with an —OH, —CF₃, —OCF₃,halogen, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 memberedheteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7membered heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,or -L¹²-OR⁸. In a related embodiment, L¹² is a bond. In other relatedembodiments, (7) and (8) may be substituted with an —OCH₃, —OCF₃, —CH₃,—CF₃, —OCH₂CH₃, halogen, or cyclopropyloxy.

R² may be: (1) unsubstituted C₃-C₇ cycloalkyl; (2) unsubstituted 3 to 7membered heterocycloalkyl; (3) unsubstituted heteroaryl; (4)unsubstituted aryl; (5) substituted C₃-C₇ cycloalkyl; (6) substituted 3to 7 membered heterocycloalkyl; (7) substituted aryl; or (8) substitutedheteroaryl. In some related embodiments, (5) and (6) are substitutedwith an oxo, —OH, —CF₃, —COOH, cyano, halogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl, -L²²-C(X³)R³, -L²²-OR⁴, -L²²-NR⁵¹R⁵², or-L²²-S(O)_(q)R⁶. X³ is ═S, ═O, or ═NR¹⁷, wherein R¹⁷ is H, —OR¹⁷¹,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl. R¹⁷¹ is H orR²¹-substituted or unsubstituted C₁-C₁₀ alkyl. The symbol q is aninteger from 0 to 2.

In other related embodiments, (7) and (8) are substituted with an —OH,—CF₃, —COOH, cyano, halogen, R²¹-substituted or unsubstituted C₁-C₁₀alkyl, R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl,-L²²-C(X³)R³, -L²²-OR⁴, -L²²-NR⁵¹R⁵², or -L²²-S(O)_(q)R⁶. L²² is a bond,unsubstituted C₁-C₁₀ alkylene or unsubstituted heteroalkylene. X³ and qare as defined above.

R³ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl, —OR³¹,or —NR³²R³³. R³² and R³³ are optionally joined with the nitrogen towhich they are attached to form an R²¹-substituted or unsubstituted 3 to7 membered heterocycloalkyl, or R²²-substituted or unsubstitutedheteroaryl.

R³¹, R³², and R³³ are independently hydrogen, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl.

R⁴, R⁵¹ and R⁵² are independently hydrogen, —CF₃, R²¹-substituted orunsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, —C(X⁴)R⁴¹, or —S(O)_(v)R⁴¹. R⁵¹ and R⁵² are optionallyjoined with the nitrogen to which they are attached to form anR²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR²²-substituted or unsubstituted heteroaryl.

X⁴ is ═S, ═O, or ═NR¹⁸, wherein R¹⁸ is R²¹-substituted or unsubstitutedC₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl. The symbol v is an integer from 0 to 2.

R⁴¹ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl, or—NR⁴¹¹R⁴¹². R⁴¹¹ and R⁴¹² are independently selected from hydrogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl. R⁴¹¹ and R⁴ are optionallyjoined with the nitrogen to which they are attached to form anR²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR²²-substituted or unsubstituted heteroaryl.

In some embodiments, R⁴¹ and R¹⁸ are optionally joined with the atoms towhich they are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁴¹¹ and R¹⁸ are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁴¹ and R⁵² are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. In otherembodiments, R⁴¹¹ and R⁵² are optionally joined with the atoms to whichthey are attached to from a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

R⁶ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl, or—NR⁶¹R⁶²R⁶¹ and R⁶² are hydrogen, R²¹-substituted or unsubstitutedC₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl. R⁶¹ and R⁶² are optionally joined with thenitrogen to which they are attached to form an R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R²²-substituted orunsubstituted heteroaryl.

R²¹ is oxo, —OH, —COOH, —CF₃, —OCF₃, —CN, amino, halogen,R²³-substituted or unsubstituted 2 to 10 membered alkyl, R²³-substitutedor unsubstituted 2 to 10 membered heteroalkyl, R²³-substituted orunsubstituted C₃-C₇ cycloalkyl, R²³-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²⁴-substituted or unsubstituted aryl, orR²⁴-substituted or unsubstituted heteroaryl.

R²² is —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, R²³-substituted orunsubstituted 2 to 10 membered alkyl, R²³-substituted or unsubstituted 2to 10 membered heteroalkyl, R²³-substituted or unsubstituted C₃-C₇cycloalkyl, R²³-substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, R²⁴-substituted or unsubstituted aryl, orR²⁴-substituted or unsubstituted heteroaryl.

R²³ is oxo, —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.

R²⁴ is —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.

In some embodiments, R² is (1), (3), (4), (5), (6), (7), or (8) (i.e.unsubstituted C₃-C₇ cycloalkyl, unsubstituted heteroaryl, unsubstitutedaryl, substituted C₃-C₇ cycloalkyl, substituted 3 to 7 memberedheterocycloalkyl, substituted aryl, or substituted heteroaryl,respectively). R² may also be (3), (4), (7), or (8). In otherembodiments, R² is (7) or (8).

In some embodiments, where R² is (7) and (8), then (7) and (8) aresubstituted with an -L²²-C(X³)R³, -L²²-OR⁴, -L²²-NR⁵¹R⁵²,-L²²-C(NH)—NR³²R³³, or -L S(O)_(q)R⁶.

In some embodiments, R³ is —NR³²R³³. X³ may be ═O or ═NR¹⁷. R⁶ may be—NR⁶¹R⁶². R⁴ may be —C(O)R⁴¹ or —S(O)_(v)R⁴¹. R⁴¹ may be —NR⁴¹¹R⁴¹².

In other embodiments where R² is (7) and (8), then (7) or (8) may besubstituted with an —OH, —CF₃, —COOH, amino, halogen, unsubstituted 2 to10 membered heteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3to 7 membered heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl, or -L 22-C(X³)R³. X³ may be ═O.

R³ may be unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 memberedheteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7membered heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,or —NR³²R³³. R³² and R³³ may independently be hydrogen, R²¹-substitutedor unsubstituted C₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl. R³² and R³³ are optionally joined with thenitrogen to which they are attached to form an R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R²²-substituted orunsubstituted heteroaryl.

In another embodiment where R² is (7) or (8), then (7) and (8) may besubstituted with unsubstituted 2 to 10 membered heteroalkyl, or-L²²-C(O)R³. L²² may be a bond. R³ may be —NR³²R³³. R³² and R³³ mayindependently be hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀alkyl, R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, or R²²-substituted or unsubstituted heteroaryl. R³²and R³³ are optionally joined with the nitrogen to which they areattached to form an R²¹-substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, or R²²-substituted or unsubstituted heteroaryl.

In some embodiments, R¹ is a substituted or unsubstituted fused ringaryl or substituted or unsubstituted fused ring heteroaryl. In otherembodiments, R² is substituted or unsubstituted indolyl, substituted orunsubstituted quinolinyl, or substituted or unsubstituted benzodioxolyl.R² may be a substituted or unsubstituted fused ring aryl or substitutedor unsubstituted fused ring heteroaryl. R¹ may be a substituted orunsubstituted indolyl, substituted or unsubstituted quinolinyl, orsubstituted or unsubstituted benzodioxolyl.

R¹ and R² may independently be a substituted or unsubstitutedhydantoinyl, substituted or unsubstituted dioxolanyl, substituted orunsubstituted dioxanyl, substituted or unsubstituted trioxanyl,substituted or unsubstituted tetrahydrothienyl, substituted orunsubstituted tetrahydrofuranyl, substituted or unsubstitutedtetrahydrothiophenyl, substituted or unsubstituted tetrahydropyranyl,substituted or unsubstituted tetrahydrothiopyranyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted morpholino,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted furanyl, substituted orunsubstituted imidazolyl, substituted or unsubstituted isoxazolyl,substituted or unsubstituted oxadiazolyl, substituted or unsubstitutedoxazolyl, substituted or unsubstituted pyridyl, substituted orunsubstituted pyrazyl, substituted or unsubstituted pyrimidyl,substituted or unsubstituted pyridazinyl, substituted or unsubstitutedthiazolyl, substituted or unsubstituted isothioazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted thienyl,substituted or unsubstituted triazinyl, substituted or unsubstitutedthiadiazolyl, or substituted or unsubstituted tetrazolyl.

In another embodiment, the compound of the present invention is any oneof the compounds of Tables 1-18 or 20, and/or of the methods 2-61 in theExamples section below.

In another aspect, the present invention provides a fused ringheterocycle kinase modulator (also referred to herein as a “compound ofthe present invention”) having the formula:

In Formula (II), L¹, L², R¹, and R² are as defined above in thediscussion of Formula (I).

In another aspect, the present invention provides a fused ringheterocycle kinase modulator (also referred to herein as a “compound ofthe present invention”) having the formula:

In Formula (III), L¹, L², R¹, and R² are as defined above in thediscussion of Formula (I).

In some embodiments, each substituted group described above in thecompounds of Formulae (I)-(III) is substituted with at least onesubstituent group. More specifically, in some embodiments, eachsubstituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,substituted alkylene, and/or substituted heteroalkylene, described abovein the compounds of Formulae (I)-(III) are substituted with at least onesubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one size-limited substituent group.Alternatively, at least one or all of these groups are substituted withat least one lower substituent group.

In other embodiments of the compounds of Formulae (I)-(III), eachsubstituted or unsubstituted alkyl is a substituted or unsubstitutedC₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is asubstituted or unsubstituted 2 to 20 membered heteroalkyl, eachsubstituted or unsubstituted cycloalkyl is a substituted orunsubstituted C₄-C₈ cycloalkyl, each substituted or unsubstitutedheterocycloalkyl is a substituted or unsubstituted 4 to 8 memberedheterocycloalkyl, each substituted or unsubstituted alkylene is asubstituted or unsubstituted C₁-C₂₀ alkylene, and/or each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20membered heteroalkylene.

Alternatively, each substituted or unsubstituted alkyl is a substitutedor unsubstituted C₁-C₈ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 8 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₈ alkylene, and/or each substitutedor unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8membered heteroalkylene.

Exemplary Syntheses

The compounds of the invention are synthesized by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate how, in principle, to gain access to thecompounds claimed under this invention and to give details on certain ofthe diverse methods available for use in assembling the compounds of theinvention. However, the discussion is not intended to define or limitthe scope of reactions or reaction sequences that are useful inpreparing the compounds of the present invention. The compounds of thisinvention may be made by the procedures and techniques disclosed in theExamples section below, as well as by known organic synthesistechniques. In Schemes 1, 2 and 3, L¹, R¹, L², and R² are as definedabove.

The key intermediates for the synthesis of 3,5-disubstituted1H-pyrazolo[3,4-b]pyridine derivatives are5-bromo-1H-pyrazolo[3,4-b]pyridine and5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine. The iodine and/or brominesubstituents on sp²-hybridized, aromatic carbon atoms present in thesebuilding blocks offer numerous synthetic possibilities forfunctionalization of either position. A great variety of such syntheticmethods exists and these procedures are generally well known andfamiliar to someone with skill in the art and include, by means ofexample and not limitation: transition metal catalyzed processes, mostnotably processes utilizing palladium, iron, nickel or copper catalysts,as well as metal-halogen exchange reactions, most notably suchprocedures introducing lithium or magnesium, and subsequent reaction ofthe transient or isolated organometallic derivative with an electrophileof suitable reactivity either directly or via transmetallation to finetune the reactivity of the organometallic species.

Using such methods, introduction of different substituents on the 3- and5-position of the 1H-pyrazolo[3,4-b]pyridine core can be accomplished byintroducing a chosen substituent at the 5-position starting from5-bromo-1H-pyrazolo[3,4-b]pyridine and subsequent halogenation,especially iodination, at position 3 of the 1H-pyrazolo[3,4-b]pyridinecore to enable the use of the aforementioned methods to introduceanother substituent of choice at that position. Alternatively, some ofthe methods outlined above may be utilized to selectively functionalize5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine at the 3-position byselectively reacting with the iodo substituent over the bromosubstituent. It is generally well known and familiar to someone withskill in the art, that a variety of palladium catalysts are known andreadily available or accessible which will exhibit higher reaction rateswith aromatic iodo substituents as compared to aromatic bromosubstituents and such catalysts may be utilized under suitableconditions to effect selective iodine substitution.

5-bromo-1H-pyrazolo[3,4-b]pyridine or a derivative containing anappropriate protecting group may also be functionalized at the3-position via various electrophilic aromatic substitution reactionsthat are generally well known and familiar to someone with skill in theart, such as FRIEDEL-CRAFTS-acylation.

The substituents introduced on either position in such fashion mayeither represent fully elaborated compounds, such as those claimed underthis invention, or they may contain functional groups, such as, forexample and without limitation, amines, carboxylic acids or esters,nitriles, olefins or halogens, either free or bearing suitableprotecting groups, which in turn may be utilized as starting material ingenerally well known synthetic transformations to synthesize compoundsthat are claimed under this invention.

Suitably functionalized pyrazolo[3,4-b]pyridine derivatives,particularly 5-bromo-1H-pyrazolo[3,4-b]pyridine and5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine, useful in synthesizingcompounds of the present invention can be prepared as outlined in Scheme1 from commercially available 5-bromo-2-fluoropyridine.5-Bromo-2-fluoropyridine can be selectively functionalized at the3-position by the generally well known selective metallation of2-fluoropyridines in a manner resembling general methods described inSchlosser, M., Organometallics in Synthesis, 2nd. ed., Wiley-VCH, 2002;Clayden, J., Organolithiums: Selectivity for Synthesis, Pergamon, 2002;and Mongin et al., Tetrahedron (2001) 57, 4059-4090. Thus, metallationmay be accomplished by treatment with a suitable, non-nucleophilicstrong base (e.g. lithium di-iso-propylamide or lithium2,2,6,6-tetramethylpiperidide) in an aprotic solvent (e.g. THF, hexanes,ether or mixtures thereof) at low temperature, typically −78° C. orbelow.

The unpurified metallated intermediate can be converted to thecorresponding 3-carbaldehyde 2 by treatment with a formylating reagentsuch as DMF, N-formyl-N-methylaniline, N-formylmorpholine,N-formylpiperidine or ethyl formate. Reaction of the carbaldehyde withhydrazine or a suitable hydrazine derivative (e.g.hydrazine-tert-butylcarbazate, or a soluble organic or inorganic saltderived from hydrazine such as hydrazine hydrochloride) either directlyor upon protection of the aldehyde using a suitable protecting group(e.g. acetal) will provide access to 5-bromo-1H-pyrazolo[3,4-b]pyridine.Introduction of a suitable group at the 3-position for furtherelaboration can be accomplished via methods generally well known in theart, such as an electrophilic aromatic substitution (e.g. bromination oriodination). Thus, the iodide 4 is accessible from 3 by treatment withsuitable reagents, such as N-iodosuccinimide, iodine monochloride oriodine, under conditions facilitating such transformation. Otherexamples of functionalization via electrophilic aromatic substitutionare, by means of example and not limitation, FRIEDEL-CRAFTS-acylationusing functionalized acyl halides such as, for example, bromoacetylchloride, acryloyl chloride or trichloroacetyl chloride in the presenceof aluminum trichloride in dichloromethane at ambient temperature orbelow. As will be appreciated by the skilled artisan, the products ofsuch reactions may be utilized as starting materials for the synthesisof certain heterocyclic compounds.

Alternatively, the metallated intermediate derived from deprotonation of5-bromo-2-fluoropyridine can be transmetallated under suitableconditions to form an organocuprate reagent (c.f. Lipshutz, B.,Organometallics in Synthesis, 2nd. ed., Wiley-VCH, 2002). Reaction ofthe cuprate generated in such fashion with an acyl halide gives accessto ketones of the general structure 5, which can be cyclized by reactionwith hydrazine or a soluble organic or inorganic salt derived fromhydrazine (e.g. hydrazine hydrochloride) to afford the corresponding3-substituted 5-bromo-1H-pyrazolo[3,4-b]pyridines of the generalstructure 6.

Elaboration of halides 3, 4 or 6 can be readily accomplished bygenerally well known methods, such as those outlined in Scheme 2 below.For example, metal catalyzed cross coupling reactions may be employedusing various known transition metal compounds (e.g. compounds derivedfrom palladium, iron or nickel). Examples of such transformations can befound in the following references: Diederich, F., Stang, P.J.—Metal-catalyzed Cross-coupling Reactions, Wiley-VCH, 1998; Beller,M., Transition Metals for Organic Synthesis, Wiley-VCH, 1998; Tsuji, J.,Palladium Reagents and Catalysts, Wiley-VCH, 1^(st). & 2^(nd) eds.,1995, 2004; Fuerstner, A., et al., J. Am. Chem. Soc. (2002) 124, 13856;and Bolm, C., et al., Chem. Rev. (2004) 104, 6217. Other useful methodsinvolve the conversion of a bromine or iodine substituent into a metalor metalloid substituent (e.g. organoboron, organolithium, organotin,organosilicon, organozinc, organocopper or organomagnesium compound)using generally well known methods (e.g. metal halogen exchange and, asappropriate or required, subsequent transmetallation using soluble andreactive compounds of boron, magnesium, zinc, tin, silicon or copper;for representative examples of such methodology see: Schlosser, M.,Organometallics in Synthesis, 2nd. ed., Wiley-VCH, 2002.).Organometallic derivatives obtained in such fashion may itself be of usein transition metal catalyzed coupling reactions with aromatic orolefinic halides or triflates, or, if sufficiently reactive, be reacteddirectly with suitable electrophiles, such as, for example, certainorganic halides, MICHAEL-acceptors, oxiranes, aziridines, aldehydes,acyl halides, or nitrites.

Selective functionalization at either the 3- or 5-position may requiredifferent strategies depending on the nature of the transformationsutilized to introduce functionalities at either position, especially thesequence of functionalization at either position. Thus, it may beadvantageous or necessary to achieve functionalization at the 3-positionprior to functionalization of the 5-position in some cases while theopposite approach may be required in other cases, depending on thenature of the specific groups to be introduced, the methods required toaccomplish such transformations, or the inherent selectivity of themethods utilized. For example, some reactants, such as for example someboronic acids or their esters that are electron deficient (e.g. containone or more electron withdrawing substituents or that representderivatives of certain heterocyclic systems) and/or contain one or moresubstituents ortho to the boron-carbon bond may require the use ofhighly active palladium catalysts (such as those mentioned in Vilar, R.,Christman, U.—Angew. Chem. (2005) 117, 370; Littke, A. F., Fu, G.—Angew.Chem. (2002) 114, 4350.) and more forcing conditions, such as highertemperatures and/or longer reaction times. Such conditions may not beconducive to achieving appreciable selectivities in reactions of5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine. Hence, in such cases, it maybe advantageous to avoid selectivity issues altogether by sequentialsubstitution of bromine in 5-bromo-1H-pyrazolo[3,4-b]pyridine,iodination at the 3-position and subsequent introduction of the secondsubstituent at position 3 utilizing the methods detailed above.Generally, when substitution of the halogen atom at either positionrequire conditions that involve highly reactive catalysts or reagentsunder conditions that generally do not favor high levels of selectivitybetween the two halogen atoms present in5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine, it may be advantageous toresort to this sequential approach.

It will also be appreciated that protection of reactive groups withinL¹, L², R¹ and/or R² as well as the pyrazolo[3,4-b]pyridine scaffold,(e.g. the proton at position 1), with a suitable protecting group may beadvantageous or required. For example it was found to be advantageous insome cross-coupling reactions to protect the nitrogen at position 1 ofthe 1H-pyrazolo[3,4-b]pyridine scaffold by introduction of either a(2-trimethylsilylethoxy)-methyl or (2-methoxy-ethoxy)methyl group atthat position. Introduction and removal of these protecting groups couldbe conveniently accomplished by methods well known in the chemicalliterature. The compounds obtained by any of the aforementioned methodsmay contain functional groups, either free or protected, that can befurther elaborated by generally well known methods.

A more detailed description of the utilization of cross-couplingprocedures in the synthesis of the compounds claimed under thisinvention is illustrated in Scheme 2: X¹ and X² are selected from, butnot limited to, halogen, boronic acid or ester, trifluoroborate salt,organomagnesium, organozinc, or organotin. With respect to theintroduction of individual residues -L¹-R¹ or -L²-R² suchtransformations, as outlined above, can be achieved via standard halogencross-coupling methodologies.

Couplings of the corresponding bromide or iodide (X¹, X²═Br, I) withsuitable reagents such as boronic acids and boronates, organoboranes,organostannanes, organozinc compounds, organomagnesium compounds,olefins or terminal alkynes (either purchased or obtained via generallywell known protocols) can be carried out in the presence of a suitabletransition metal catalyst (e.g. palladium compounds). The coupling mayoptionally be performed in the presence of ligands such as phosphines,diphosphines, Arduengo-type heterocyclic carbenes or arsines. Organic orinorganic bases (e.g. tertiary or secondary amines, alkaline carbonates,bicarbonates or phosphate) and/or other well known additives (e.g.lithium chloride, copper halides or silver salts) may be utilized toassist or accelerate such transformations.

These cross coupling reactions may be carried out in suitable solventssuch as THF, dioxane, dimethoxyethane, diglyme, dichloromethane,dichloroethane, acetonitrile, DMF, N-methylpyrrolidone, water, ormixtures of thereof at temperatures ranging from 25° C. to 200° C.using. The temperature may optionally be maintained with heating,conventional heating or microwave irradiation. In the case of the3-iodo-5-bromo-1H-pyrazolo[3,4-b]pyridine, the selective or preferentialsubstitution of the iodo substituent over the bromo substituent ispossible under generally less forcing conditions, such as lowertemperature and shorter reaction times using a suitable transition metalcatalyst. Selective functionalizations of di- or oligohalogen compoundsby means of transition metal catalyzed transformations are wellprecedented in the chemical literature: see for example Ji, J., et al.Org. Lett (2003) 5, 4611; Bach, T., et al., J. Org. Chem. (2002) 67,5789, Adamczyk, M. et. al., Tetrahedron (2003) 59, 8129.

This methodology may be extended to the incorporation of non-carbonbased nucleophiles (e.g. alcohols, thiols, primary or secondary amines)that may optionally contain suitable protecting groups of alcohols,thiols or amines. Examples of such groups can be found in Greene, T., etal., Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons,1999. Exemplary methods of protection are described in Ley, S., et al.,Angew. Chem. (2003) 115, 5558; Wolfe, J., et al., Acc. Chem. Res. (1998)31, 805; Hartwig, Acc. Chem. Res. (1998) 31, 852; Navarro, O., et al.,J. Org. Chem. (2004) 69, 3173, Ji, J., et al., Org. Lett (2003) 5, 4611.The compounds obtained by such methods can be further elaborated by wellknown methods to obtain other compounds of the present invention.

In some cases it may be advantageous to achieve cross-couplings tocarbon or non-carbon atoms by first converting the respective halogenderivative into the corresponding organometallic derivative (e.g., aboronic acid or ester, trifluoroborate salt, organomagnesium, organozincor organotin compound). Such compounds are accessible by means ofsubstituting the halide moiety with an appropriate metal or metalloid.Any functional groups present (e.g. the ring nitrogen in position 1 ofthe pyrazolo[3,4-b]pyridine), may need to be protected by a suitableprotecting group (“PG”). See Greene, et al, 1999.

Introduction of such metals or metalloids can be achieved by generallywell-known methods, such as metallation using metals or a metal-halogenexchange reaction. Useful metals for metallation include alkaline oralkaline earth metals or activated forms of such metals. Suitablereagents for use in metal-halogen exchange reactions includeorganolithium or organomagnesium compounds (e.g. n-butyllithium,tert-butyllithium or iso-propylmagnesium chloride or bromide).Subsequent transmetalation reactions of the organometallic intermediatemay be performed as needed with a suitable soluble and reactive metalcompound such as magnesium chloride, magnesium bromide, tri-n-butyltinchloride, trimethyltin chloride, trimethyl borate, triethyl borate,tri-iso-propyl borate, zinc triflate or zinc chloride. Introduction of aboronic acid pinacol ester can be conveniently achieved by reacting thehalogen derivative directly with bis(pinacolato)diboron in the presenceof dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) andsuitable bases (e.g. potassium or sodium acetate) in solvents such asDMSO, DMF, DMA or N-methylpyrrolidone at temperatures ranging from80-160° C. Conventional heating or microwave irradiation may be employedto maintain the appropriate temperature (for literature precedent ofsimilar transformations, see Ishiyama, T., et al., J. Org. Chem. (1995)60, 7508.).

Methods for conversion of the boronic acid pinacol ester obtained bythis method into other boronic acid derivatives such as boronic acids,boronates, or trifluoroborate salts are generally well known. As will beapparent to the skilled artisan, such organometallic derivatives may beutilized in cross-coupling reactions similar to those described above inthe case of halogen containing derivatives of pyrazolo[3,4-b]pyridine.Such couplings can be effected utilizing suitable coupling partners,such as aromatic, heteroaromatic halides or olefinic reagents underconditions identical or evidently similar and/or related to the methodsdescribed above.

Other methods may utilize the reactivity of organometallic derivativesgenerated from halogen containing derivatives of pyrazolo[3,4-b]pyridineby any of the methods described above. For example, derivativescontaining alkaline or alkaline earth metals (e.g. organolithium,organomagnesium or organozinc compounds) may be employed in directcouplings to a range of other electrophilic coupling partners such as,for example, activated olefins (MICHAEL-acceptors), aldehydes, nitrites,aromatic nitro compounds, carboxylic acid derivatives, oxiranes,aziridines, organic disulfides or organic halides. Such transformationsare generally well known in the art (for reactions with aromatic nitrocompounds, see for example Sapountzis, I., et al., J. Am. Chem. Soc.(2002) 124, 9390.).

The synthetic strategies utilized to access 3,5-disubstituted1H-pyrrolo[2,3-b]pyrazine derivatives are closely related to thestrategies described above for 1H-pyrazolo[3,4-b]pyridine derivatives,with the main difference relating to the synthesis of the1H-pyrrolo[2,3-b]pyrazine scaffold itself. The key intermediatesutilized are 3-substituted 5-iodo-1H-pyrrolo[2,3-b]pyrazine derivativesand 5-bromo-1H-pyrrolo[2,3-b]pyrazine itself.

The general synthetic strategies to access 3,5-disubstituted1H-pyrazolo[3,4-b]pyridine derivatives from5-bromo-1H-pyrazolo[3,4-b]pyridine outlined above will also pertain toaccessing 3,5-disubstituted 1H-pyrrolo[2,3-b]pyrazine derivatives from5-bromo-1H-pyrrolo[2,3-b]pyrazine and 3-substituted5-iodo-1H-pyrrolo[2,3-b]pyrazine derivatives. However, the exactconditions for otherwise similar or identical transformations may verywell be different for 1H-pyrrolo[2,3-b]pyrazine derivatives andoptimization depending on the scaffold utilized may be required.

5-Bromo-1H-pyrrolo[2,3-b]pyrazine is accessible via regioselectiveSONOGASHIRA-coupling of 3-amino-2,6-dibromo-pyrazine withtrimethylsilylacetylene (see Adamczyk, M., et al.—Tetrahedron (2003) 59,8129.), N-acylation, and subsequent cyclization using -n-butylammoniumfluoride (for precedent of this reaction please see WO2004/032874A2).Starting from commercially available 3-amino-2,6-dibromo-pyrazine,5-bromo-3-trimethylsilanylethynyl-pyrazin-2-ylamine can be obtained byreaction with trimethylsilylacetylene in the presence of a palladiumcatalyst, such as tetrakis(triphenylphosphino)palladium(0) and acatalytic amount of a copper co-catalyst, such as copper(I)-iodide in amixture of DMF and a basic tertiary organic amine, such as triethylamineat elevated temperatures. Acetylation with acetyl chloride in pyridineat 20-60° C. gives access toN-(5-bromo-3-trimethylsilanylethynyl-pyrazin-2-yl)-acetamide andsubsequent cyclization with tetra-n-butylammonium fluoride in THF underreflux affords 5-bromo-1H-pyrrolo[2,3-b]pyrazine.

Introduction of a suitable group at the 3-position for furtherelaboration can be accomplished via methods generally well known in theart, such as an electrophilic aromatic substitution (e.g. bromination oriodination). Thus, 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyrazine isaccessible from 5-bromo-1H-pyrrolo[2,3-b]pyrazine by treatment withsuitable reagents, such as N-iodosuccinimide, iodine monochloride oriodine, under conditions facilitating such transformation.

Other examples of functionalization via electrophilic aromaticsubstitution are, by means of example and not limitation,FRIEDEL-CRAFTS-acylation using functionalized acyl halides such as, forexample, bromoacetyl chloride, acryloyl chloride or trichloroacetylchloride in the presence of aluminum trichloride in dichloromethane atambient temperature or below. As will be appreciated by the skilledartisan, the products of such reactions either represent compoundsclaimed under this invention or may be utilized as starting materialsfor the synthesis of such compounds, most notably certain heterocycliccompounds.

Further elaboration of halide D (X²═Br, I) as well as selectivesequential substitution of both halogen substituents in5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyrazine can be readily accomplished bygenerally well known methods, such as, for example, sequential metalcatalyzed cross coupling reactions may be employed using various knowntransition metal compounds (e.g. compounds derived from palladium, ironor nickel). Examples of such transformations can be found in thefollowing references: Diederich, F., Stang, P. J.—Metal-catalyzedCross-coupling Reactions, Wiley-VCH, 1998; Beller, M., Transition Metalsfor Organic Synthesis, Wiley-VCH, 1998; Tsuji, J., Palladium Reagentsand Catalysts, Wiley-VCH, 1^(st). & 2^(nd) eds., 1995, 2004; Fuerstner,A., et al., J. Am. Chem. Soc. (2002) 124, 13856; and Bolm, C., et al.,Chem. Rev. (2004) 104, 6217. The general methods known in the chemicalliterature and familiar to someone with skill in the art are essentiallythe same methods as those described above for similar or identicaltransformations utilizing 1H-pyrazolo[3,4-b]pyridine derivatives.

As was discussed for 1H-pyrazolo[3,4-b]pyridines the skilled artisanwill appreciate that selective functionalization at either the 3- or5-position may require different strategies depending on the nature ofthe transformations utilized to introduce functionalities at eitherposition, especially the sequence of functionalization at eitherposition. Thus, it may be advantageous or necessary to achievefunctionalization at the 3-position prior to functionalization of the5-position in some cases while the opposite approach may be required inother cases, depending on the nature of the specific groups to beintroduced, the methods required to accomplish such transformations, orthe inherent selectivity of the methods utilized.

In the case of the 3-iodo-5-bromo-1H-pyrrolo[2,3-b]pyrazine, theselective or preferential substitution of the iodo substituent over thebromo substituent is possible under generally less forcing conditions,such as lower temperature and shorter reaction times using a suitabletransition metal catalyst. Selective functionalizations of di- oroligohalogen compounds by means of transition metal catalyzedtransformations are well precedented in the chemical literature: see forexample Ji, J., et al. Org. Lett (2003) 5, 4611; Bach, T., et al., J.Org. Chem (2002) 67, 5789, Adamczyk, M. et. al., Tetrahedron (2003) 59,8129.

In the case of halide D (X²═Br, I) other useful methods may involve theconversion of a bromine or iodine substituent into a metal or metalloidsubstituent (e.g. organoboron, organoithium, organotin, organosilicon,organozinc, organocopper or organomagnesium compound) using generallywell known methods (e.g. metal halogen exchange and, as appropriate orrequired, subsequent transmetallation using soluble and reactivecompounds of boron, magnesium, zinc, tin, silicon or copper; forrepresentative examples of such methodology see: Schlosser, M.,Organometallics in Synthesis, 2nd. ed., Wiley-VCH, 2002). Organometallicderivatives obtained in such fashion may itself be of use in transitionmetal catalyzed coupling reactions with aromatic or olefinic halides ortriflates, or, if sufficiently reactive, be reacted directly withsuitable electrophiles, such as, for example, certain organic halides,MICHAEL-acceptors, oxiranes, aziridines, aldehydes, acyl halides, ornitriles. Again, the general methods known in the chemical literatureare essentially the same as those described above for similar oridentical transformations utilizing 1H-pyrazolo[3,4-b]pyridinederivatives.

In certain such transformations, it may be advantageous or required tointroduce one or more suitable protecting groups, in order totemporarily substitute acidic protons, such as, for example, thehydrogen atoms attached to nitrogen or oxygen, as needed, and inparticular the hydrogen atom in position 1 of the1H-pyrrolo[2,3-b]pyrazine scaffold, by methods well known in thechemical literature (cf. T. W. Greene, P. G. M. Wuts —Protective Groupsin Organic Synthesis, 3rd ed., John Wiley & Sons, 1999).

The cross-coupling methodology described above may be extended to theincorporation of non-carbon based nucleophiles (e.g. alcohols, thiols,primary or secondary amines) that may optionally contain suitableprotecting groups of alcohols, thiols or amines. Examples of such groupscan be found in Greene, T., et al., Protective Groups in OrganicSynthesis, 3rd ed., John Wiley & Sons, 1999. Exemplary methods ofprotection are described in Ley, S., et al., Angew. Chem. (2003) 115,5558; Wolfe, J., et al., Acc. Chem. Res. (1998) 31, 805; Hartwig, Acc.Chem. Res. (1998) 31, 852; Navarro, O., et al., J. Org. Chem. (2004) 69,3173, Ji, J., et al., Org. Lett (2003) 5, 4611. The compounds obtainedby such methods can be further elaborated by well known methods toobtain other compounds of the present invention. In some cases, directsubstitution of the 5-iodo or 5-bromo substituent in1H-pyrrolo[2,3-b]pyrazine with an amine, alcohol or thiol may besuccessfully accomplished at ambient or elevated temperatures in thepresence of weak acids, such as, for example, acetic acid, or a strong,non-nucleophilic base, such as, for example, sodium hydride either inneat amine, alcohol or thiol, respectively or in a suitable aproticsolvent, such as, for example, DMF, NMP, DMSO, or acetonitrile.

An alternative method for the synthesis of 3,5-disubstituted1H-pyrrolo[2,3-b]pyrazine derivatives was developed, starting frommethyl 2-amino-3-pyrazinecarboxylate, incorporation of an iodine atom onthe 5-position to give methyl 2-amino-5-iodo-3-pyrazinecarboxylate canbe achieved by various known methods, such as reaction withN-iodosuccinimide in ethanol at reflux. The halogenated ester obtainedby such means may then be hydrolized by standard methods. For example,treatment with lithium hydroxide in THF-water mixtures at ambienttemperature affords the corresponding acid.

Synthesis of a ketone intermediate B can be achieved by treating thecorresponding WEINREB-amide A (3-amino-6-iodo-pyrazine-2-carboxylic acidmethoxy-methyl-amide) or its hydrochloride salt with a suitableorganometallic species, for example, using an organomagnesium ororganolithium compound. (for examples of the use ofN-methoxy-N-methylamides (Weinreb Amides) in ketone synthesis, see S.Nam, S. M. Weinreb—Tetrahedron Lett. 1981, 22, 3815.)3-Amino-6-iodo-pyrazine-2-carboxylic acid methoxy-methyl-amide (A) isaccessible by condensation of the parent acid with N,O-dimethylhydroxylamine using standard methods for amide-formation,either by prior activation of the acid or in situ or via directcondensation. Methods and reagents for both transformations aredescribed in the chemical literature and well known to someone skilledin the art, such as in the case of direct methods using suitablecoupling reagents such as, but not limited to, PyBOP, HBTU or HATU.

The organometallic reagents required for the introduction of a ketoneresidue L¹R¹ in B can be obtained either commercially or synthesized byvarious methods described in the literature, such as, but not limited tothe GRIGNARD-reaction of organic chlorides, bromides, or iodides, withmagnesium (cf. J. March—Advanced Organic Chemistry, 3rd ed., John Wiley& Sons, 1992), metal-halogen exchange reactions of organic bromides oriodides using suitable organolithium or organomagnesium compounds suchas, but not limited to, n-butyllithium, tert-butyllithium oriso-propylmagnesium chloride or bromide (e.g. J. Clayden—Organolithiums:Selectivity for Synthesis, Pergamon, 2002; A. Boudier, L. O. Bromm, M.Lotz, P. Knochel—Angew. Chem. Int. Ed. (2000) 39, 4414.) ordeprotonation of sufficiently acidic compounds, such as for examplepyrimidines, pyrazines, 2-chloro- or 2-fluoropyridines using a suitablebase, such as for example lithium N,N-diisopropylamide or lithium2,2,6,6-tetramethylpiperidide (cf. J. Clayden—Organolithiums:Selectivity for Synthesis, Pergamon, 2002; A. Turck, N. Plé, F. Mongin,G. Quéguiner—Tetrahedron (2001) 57, 4489; F. Mongin, G.Quéguiner—Tetrahedron (2001) 57, 4059). In certain such transformations,it may be advantageous or required to introduce one or more suitableprotecting groups, in order to temporarily substitute acidic protons(e.g. the hydrogen atoms attached to nitrogen or oxygen) as needed, bymethods well known in the chemical literature (cf. T. W. Greene, P. G.M. Wuts—Protective Groups in Organic Synthesis, 3rd ed., John Wiley &Sons, 1999).

Conversion of the ketone intermediate B to the methoxyvinyl derivative Ccan be achieved by several known methods but is most convenientlycarried out via a WITTIG-reaction (cf. B. E. Maryanoff, A. B.Reitz—Chem. Rev. (1989) 89, 863) using an ylid generated fromcommercially available methoxymethyltriphenylphosphonium chloride and asuitable base, for example, but not limited to, a strong organometallicbase such as, but not limited to, a non-nucleophilic amide such as thelithium, sodium or potassium salt of bis(trimethylsilyl) amine.

Subsequent cyclization of the resulting olefin C, which can be utilizedin either the E- or Z-form or a mixture of these both forms, can beachieved under general acid catalysis conditions to afford 3-substituted1H-5-iodo-pyrrolo[2,3-b]pyrazines. Such methods may utilize stronginorganic or organic acids, such as sulfuric acid, perchloric acid,hydrochloric acid, trifluoromethanesulfonic acid or trifluoroacetic acidin suitable solvents (e.g. THF, dioxane, diethyl ether, dimethoxyethane,diglyme, dichloromethane, dichloroethane or chloroform, water, methanol,or ethanol, or mixtures thereof) at temperatures ranging from 0° C. to160° C. A similar cyclization has been described by Sakamoto et al.,Heterocycles (1992), 34(12), 2379-84. There the authors describe theconversion of 2-nitro-3-(2-ethoxyvinyl)pyridine to the parentpyrrolo[2,3-b]pyridine. Formation of the vinyl group was reported to beachieved via a STILLE-coupling of the 3-bromo analog withtributyl-2-ethoxyvinylstannane.

The utility of 3-substituted 1H-5-iodo-pyrrolo[2,3-b]pyrazines in thesynthesis of compounds claimed under this invention will be obvious tosomeone skilled in the art based on the methods described above. One ofskill will immediately understand that the synthetic methods describedherein, including the Examples section below, may be used and/orelaborated to obtain the compounds of Formulae (I), (II), and/or (III).

A. Protecting Groups

The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed.John Wiley & Sons (1999). It may be advantageous, where differentprotecting groups are employed, that each (different) protective groupbe removable by a different means. Protective groups that are cleavedunder totally disparate reaction conditions allow differential removalof such protecting groups. For example, protective groups can be removedby acid, base, and hydrogenolysis. Groups such as trityl,dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile andmay be used to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and Fmoc groups, which are base labile. Carboxylicacid and hydroxy reactive moieties may be blocked with base labilegroups such as, without limitation, methyl, ethyl, and acetyl in thepresence of amines blocked with acid labile groups such as tert-butylcarbamate or with carbamates that are both acid and base stable buthydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking/protecting groups include, but are not limited to thefollowing moieties:

II. Methods of Inhibiting Kinases

In another aspect, the present invention provides methods of modulatingprotein kinase activity using the fused ring heterocycle kinasemodulators of the present invention. The term “modulating kinaseactivity,” as used herein, means that the activity of the protein kinaseis increased or decreased when contacted with a fused ring heterocyclekinase modulator of the present invention relative to the activity inthe absence of the fused ring heterocycle kinase modulator. Therefore,the present invention provides a method of modulating protein kinaseactivity by contacting the protein kinase with a fused ring heterocyclekinase modulator of the present invention (e.g. the compounds of any oneof Formulae (I)-(III)).

In an exemplary embodiment, the fused ring heterocycle kinase modulatorinhibits kinase activity. The term “inhibit,” as used herein inreference to kinase activity, means that the kinase activity isdecreased when contacted with a fused ring heterocycle kinase modulatorrelative to the activity in the absence of the fused ring heterocyclekinase modulator. Therefore, the present invention further provides amethod of inhibiting protein kinase activity by contacting the proteinkinase with a fused ring heterocycle kinase modulator of the presentinvention.

In certain embodiments, the protein kinase is a protein tyrosine kinase.A protein tyrosine kinase, as used herein, refers to an enzyme thatcatalyzes the phosphorylation of tyrosine residues in proteins with aphosphate donors (e.g. a nucleotide phosphate donor such as ATP).Protein tyrosine kinases include, for example, Abelson tyrosine kinases(“Abl”) (e.g. c-Abl and v-Abl), Ron receptor tyrosine kinases (“RON”),Met receptor tyrosine kinases (“MET”), Fms-like tyrosine kinases (“FLT”)(e.g. FLT3), src-family tyrosine kinases (e.g. lyn, CSK), andp21-activated kinase-4 (“PAK”), FLT3, aurora kinases, B-lymphoidtyrosine kinases (“Blk”), cyclin-dependent kinases (“CDK”) (e.g. CDK1and CDK5), src-family related protein tyrosine kinases (e.g. Fynkinase), glycogen synthase kinases (“GSK”) (e.g. GSK3α and GSK3β),lymphocyte protein tyrosine kinases (“Lck”), ribosomal S6 kinases (e.g.Rsk1, Rsk2, and Rsk3), sperm tyrosine kinases (e.g. Yes), and subtypesand homologs thereof exhibiting tyrosine kinase activity. In certainembodiments, the protein tyrosine kinase is Abl, RON, MET, PAK, or FLT3.In other embodiments, the protein tyrosine kinase is a FLT3 or Ablfamily member.

In some embodiments, the kinase is selected from Abelson tyrosinekinase, Ron receptor tyrosine kinase, Met receptor tyrosine kinase,Fms-like tyrosine kinase-3, Aurora kinases, p21-activated kinase-4, and3-phosphoinositide-dependent kinase-1.

In another embodiment, the kinase is a mutant kinase, such as a mutantBcr-Abl kinase, FLT3 kinase or aurora kinases. Useful mutant Bcr-Ablkinases include those having at least one of the following clinicallyisolated mutations: M244V, L248V, G250E, G250A, Q252H, Q252R, Y253F,Y253H, E255K, E255V, D276G, F311L, T3151, T315N, T315A, F317V, F317L,M343T, M351T, E355G, F359A, F359V, V379I, F382L, L387M, H396P, H396R,S417Y, E459K and F486S. In some embodiments, the mutant Abl kinase has aT315I mutation. The numbering system denoting the position of the aminoacid mutation above the well known wild-type ABL numbering according toABL exon Ia. See Deininger, M., et al., Blood 105(7), 2640 (2005). Thenumbering system is reproduced in FIG. 1. In some embodiments, themutant Bcr-Abl kinase includes at least one of the mutations listedabove and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the sequence of FIG. 1. In some embodiments, themutant Bcr-Abl kinase includes at least one of the mutations listedabove, has a sequence identity to FIG. 1 as discussed above, andincludes at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1100 amino acids.

In some embodiments, the kinase is homologous to a known kinase (alsoreferred to herein as a “homologous kinase”). Compounds and compositionsuseful for inhibiting the biological activity of homologous kinases maybe initially screened, for example, in binding assays. Homologousenzymes comprise an amino acid sequence of the same length that is atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%identical to the amino acid sequence of full length known kinase, or70%, 80%, or 90% homology to the known kinase active domains. Homologymay be determined using, for example, a PSI BLAST search, such as, butnot limited to that described in Altschul, et al., Nuc. Acids Rec.25:3389-3402 (1997). In certain embodiments, at least 50%, or at least70% of the sequence is aligned in this analysis. Other tools forperforming the alignment include, for example, DbClustal and ESPript,which may be used to generate the PostScript version of the alignment.See Thompson et al., Nucleic Acids Research, 28:2919-26, 2000; Gouet, etal., Bioinformatics, 15:305-08 (1999). Homologs may, for example, have aBLAST E-value of 1×10⁻⁶ over at least 100 amino acids (Altschul et al.,Nucleic Acids Res., 25:3389-402 (1997) with FLT3, Abl, or another knownkinase, or any functional domain of FLT3, Abl, or another known kinase.

Homology may also be determined by comparing the active site bindingpocket of the enzyme with the active site binding pockets of a knownkinase. For example, in homologous enzymes, at least 50%, 60%, 70%, 80%,or 90% of the amino acids of the molecule or homolog have amino acidstructural coordinates of a domain comparable in size to the kinasedomain that have a root mean square deviation of the alpha carbon atomsof up to about 1.5 Å, about 1.25 Å, about 1 Å, about 0.75 Å, about 0.5Å, and or about 0.25 Å.

The compounds and compositions of the present invention are useful forinhibiting kinase activity and also for inhibiting other enzymes thatbind ATP. They are thus useful for the treatment of diseases anddisorders that may be alleviated by inhibiting such ATP-binding enzymeactivity. Methods of determining such ATP binding enzymes include thoseknown to those of skill in the art, those discussed herein relating toselecting homologous enzymes, and by the use of the database PROSITE,where enzymes containing signatures, sequence patterns, motifs, orprofiles of protein families or domains may be identified.

The compounds of the present invention, and their derivatives, may alsobe used as kinase-binding agents. As binding agents, such compounds andderivatives may be bound to a stable resin as a tethered substrate foraffinity chromatography applications. The compounds of this invention,and their derivatives, may also be modified (e.g., radiolabelled oraffinity labeled, etc.) in order to utilize them in the investigation ofenzyme or polypeptide characterization, structure, and/or function.

In an exemplary embodiment, the fused ring heterocycle kinase modulatorof the present invention is a kinase inhibitor. In some embodiments, thekinase inhibitor has an IC₅₀ of inhibition constant (K_(i)) of less than1 micromolar. In another embodiment, the kinase inhibitor has an IC₅₀ orinhibition constant (K_(i)) of less than 500 micromolar. In anotherembodiment, the kinase inhibitor has an IC₅₀ or K_(i) of less than 10micromolar. In another embodiment, the kinase inhibitor has an IC₅₀ orK_(i) of less than 1 micromolar. In another embodiment, the kinaseinhibitor has an IC₅₀ or K_(i) of less than 500 nanomolar. In anotherembodiment, the kinase inhibitor has an IC₅₀ or K_(i) of less than 10nanomolar. In another embodiment, the kinase inhibitor has an IC₅₀ orK_(i) of less than 1 nanomolar.

III. Methods of Treatment

In another aspect, the present invention provides methods of treating adisease mediated by kinase activity (kinase-mediated disease ordisorder) in a subject (e.g. mammals, such as humans). By“kinase-mediated” or “kinase-associated” diseases is meant diseases inwhich the disease or symptom can be alleviated by inhibiting kinaseactivity (e.g. where the kinase is involved in signaling, mediation,modulation, or regulation of the disease process). By “diseases” ismeant diseases, or disease symptoms. The method includes administeringto the subject an effective amount of a fused ring heterocycle kinasemodulator of the present invention (e.g. the compounds of any one ofFormulae (I)-(III)).

Examples of kinase associated diseases include cancer (e.g. leukemia,tumors, and metastases), allergy, asthma, obesity, inflammation (e.g.inflammatory diseases such as inflammatory airways disease),hematological disorders, obstructive airways disease, asthma, autoimmunediseases, metabolic diseases, infection (e.g. bacterial, viral, yeast,fungal), CNS diseases, brain tumors, degenerative neural diseases,cardiovascular diseases, and diseases associated with angiogenesis,neovascularization, and vasculogenesis. In an exemplary embodiment, thecompounds are useful for treating cancer, including leukemia, and otherdiseases or disorders involving abnormal cell proliferation, such asmyeloproliferative disorders.

More specific examples of cancers treated with the compounds of thepresent invention include breast cancer, lung cancer, melanoma,colorectal cancer, bladder cancer, ovarian cancer, prostate cancer,renal cancer, squamous cell cancer, glioblastoma, pancreatic cancer,Kaposi's sarcoma, multiple myeloma, and leukemia (e.g. myeloid, chronicmyeloid, acute lymphoblastic, chronic lymphoblastic, Hodgkins, and otherleukemias and hematological cancers).

Other specific examples of diseases or disorders for which treatment bythe compounds or compositions of the invention are useful for treatmentor prevention include, but are not limited to transplant rejection (forexample, kidney, liver, heart, lung, islet cells, pancreas, bone marrow,cornea, small bowel, skin allografts or xenografts and othertransplants), graft vs. host disease, osteoarthritis, rheumatoidarthritis, multiple sclerosis, diabetes, diabetic retinopathy,inflammatory bowel disease (for example, Crohn's disease, ulcerativecolitis, and other bowel diseases), renal disease, cachexia, septicshock, lupus, myasthenia gravis, psoriasis, dermatitis, eczema,seborrhea, Alzheimer's disease, Parkinson's disease, stem cellprotection during chemotherapy, ex vivo selection or ex vivo purging forautologous or allogeneic bone marrow transplantation, ocular disease,retinopathies (for example, macular degeneration, diabetic retinopathy,and other retinopathies), corneal disease, glaucoma, infections (forexample bacterial, viral, or fungal), heart disease, including, but notlimited to, restenosis.

IV. Assays

The compounds of the present invention may be easily assayed todetermine their ability to modulate protein kinases, bind proteinkinases, and/or prevent cell growth or proliferation. Some examples ofuseful assays are presented below.

A. Kinase Inhibition and Binding Assays

Inhibition of various kinases is measured by methods known to those ofordinary skill in the art, such as the various methods presented herein,and those discussed in the Upstate KinaseProfiler Assay Protocols June2003 publication.

For example, where in vitro assays are performed, the kinase istypically diluted to the appropriate concentration to form a kinasesolution. A kinase substrate and phosphate donor, such as ATP, is addedto the kinase solution. The kinase is allowed to transfer a phosphate tothe kinase substrate to form a phosphorylated substrate. The formationof a phosphorylated substrate may be detected directly by anyappropriate means, such as radioactivity (e.g. [γ-³²P-ATP]), or the useof detectable secondary antibodies (e.g. ELISA). Alternatively, theformation of a phosphorylated substrate may be detected using anyappropriate technique, such as the detection of ATP concentration (e.g.Kinase-Glo® assay system (Promega)). Kinase inhibitors are identified bydetecting the formation of a phosphorylated substrate in the presenceand absence of a test compound (see Examples section below).

The ability of the compound to inhibit a kinase in a cell may also beassayed using methods well known in the art. For example, cellscontaining a kinase may be contacted with an activating agent (such as agrowth factor) that activates the kinase. The amount of intracellularphosphorylated substrate formed in the absence and the presence of thetest compound may be determined by lysing the cells and detecting thepresence phosphorylated substrate by any appropriate method (e.g.ELISA). Where the amount of phosphorylated substrate produced in thepresence of the test compound is decreased relative to the amountproduced in the absence of the test compound, kinase inhibition isindicated. More detailed cellular kinase assays are discussed in theExamples section below.

To measure the binding of a compound to a kinase, any method known tothose of ordinary skill in the art may be used. For example, a test kitmanufactured by Discoverx (Fremont, Calif.), ED-Staurosporine NSIP™Enzyme Binding Assay Kit (see U.S. Pat. No. 5,643,734) may be used.Kinase activity may also be assayed as in U.S. Pat. No. 6,589,950,issued Jul. 8, 2003.

Suitable kinase inhibitors may be selected from the compounds of theinvention through protein crystallographic screening, as disclosed in,for example Antonysamy, et al., PCT Publication No. WO03087816A1, whichis incorporate herein by reference in its entirety for all purposes.

The compounds of the present invention may be computationally screenedto assay and visualize their ability to bind to and/or inhibit variouskinases. The structure may be computationally screened with a pluralityof compounds of the present invention to determine their ability to bindto a kinase at various sites. Such compounds can be used as targets orleads in medicinal chemistry efforts to identify, for example,inhibitors of potential therapeutic importance (Travis, Science,262:1374, 1993). The three dimensional structures of such compounds maybe superimposed on a three dimensional representation of kinases or anactive site or binding pocket thereof to assess whether the compoundfits spatially into the representation and hence the protein. In thisscreening, the quality of fit of such entities or compounds to thebinding pocket may be judged either by shape complementarity or byestimated interaction energy (Meng, et al., J. Comp. Chem. 13:505-24,1992).

The screening of compounds of the present invention that bind to and/ormodulate kinases (e.g. inhibit or activate kinases) according to thisinvention generally involves consideration of two factors. First, thecompound must be capable of physically and structurally associating,either covalently or non-covalently with kinases. For example, covalentinteractions may be important for designing irreversible or suicideinhibitors of a protein. Non-covalent molecular interactions importantin the association of kinases with the compound include hydrogenbonding, ionic interactions, van der Waals, and hydrophobicinteractions. Second, the compound must be able to assume a conformationand orientation in relation to the binding pocket, that allows it toassociate with kinases. Although certain portions of the compound willnot directly participate in this association with kinases, thoseportions may still influence the overall conformation of the moleculeand may have a significant impact on potency. Conformationalrequirements include the overall three-dimensional structure andorientation of the chemical group or compound in relation to all or aportion of the binding pocket, or the spacing between functional groupsof a compound comprising several chemical groups that directly interactwith kinases.

Docking programs described herein, such as, for example, DOCK, or GOLD,are used to identify compounds that bind to the active site and/orbinding pocket. Compounds may be screened against more than one bindingpocket of the protein structure, or more than one set of coordinates forthe same protein, taking into account different molecular dynamicconformations of the protein. Consensus scoring may then be used toidentify the compounds that are the best fit for the protein (Charifson,P. S. et al., J. Med. Chem. 42: 5100-9 (1999)). Data obtained from morethan one protein molecule structure may also be scored according to themethods described in Klingler et al., U.S. Utility Application, filedMay 3, 2002, entitled “Computer Systems and Methods for VirtualScreening of Compounds.” Compounds having the best fit are then obtainedfrom the producer of the chemical library, or synthesized, and used inbinding assays and bioassays.

Computer modeling techniques may be used to assess the potentialmodulating or binding effect of a chemical compound on kinases. Ifcomputer modeling indicates a strong interaction, the molecule may thenbe synthesized and tested for its ability to bind to kinases and affect(by inhibiting or activating) its activity.

Modulating or other binding compounds of kinases may be computationallyevaluated by means of a series of steps in which chemical groups orfragments are screened and selected for their ability to associate withthe individual binding pockets or other areas of kinases. This processmay begin by visual inspection of, for example, the active site on thecomputer screen based on the kinases coordinates. Selected fragments orchemical groups may then be positioned in a variety of orientations, ordocked, within an individual binding pocket of kinases (Blaney, J. M.and Dixon, J. S., Perspectives in Drug Discovery and Design, 1:301,1993). Manual docking may be accomplished using software such as InsightII (Accelrys, San Diego, Calif.) MOE (Chemical Computing Group, Inc.,Montreal, Quebec, Canada); and SYBYL (Tripos, Inc., St. Louis, Mo.,1992), followed by energy minimization and/or molecular dynamics withstandard molecular mechanics force fields, such as CHARMM (Brooks, etal., J. Comp. Chem. 4:187-217, 1983), AMBER (Weiner, et al., J. Am.Chem. Soc. 106: 765-84, 1984) and C² MMFF (Merck Molecular Force Field;Accelrys, San Diego, Calif.). More automated docking may be accomplishedby using programs such as DOCK (Kuntz et al., J. Mol. Biol., 161:269-88,1982; DOCK is available from University of California, San Francisco,Calif.); AUTODOCK (Goodsell & Olsen, Proteins: Structure, Function, andGenetics 8:195-202, 1990; AUTODOCK is available from Scripps ResearchInstitute, La Jolla, Calif.); GOLD (Cambridge Crystallographic DataCentre (CCDC); Jones et al., J. Mol. Biol. 245:43-53, 1995); and FLEXX(Tripos, St. Louis, Mo.; Rarey, M., et al., J. Mol. Biol. 261:470-89,1996). Other appropriate programs are described in, for example,Halperin, et al.

During selection of compounds by the above methods, the efficiency withwhich that compound may bind to kinases may be tested and optimized bycomputational evaluation. For example, a compound that has been designedor selected to function as a kinases inhibitor may occupy a volume notoverlapping the volume occupied by the active site residues when thenative substrate is bound, however, those of ordinary skill in the artwill recognize that there is some flexibility, allowing forrearrangement of the main chains and the side chains. In addition, oneof ordinary skill may design compounds that could exploit proteinrearrangement upon binding, such as, for example, resulting in aninduced fit. An effective kinase inhibitor may demonstrate a relativelysmall difference in energy between its bound and free states (i.e., itmust have a small deformation energy of binding and/or lowconformational strain upon binding). Thus, the most efficient kinaseinhibitors should, for example, be designed with a deformation energy ofbinding of not greater than 10 kcal/mol, not greater than 7 kcal/mol,not greater than 5 kcal/mol, or not greater than 2 kcal/mol. Kinaseinhibitors may interact with the protein in more than one conformationthat is similar in overall binding energy. In those cases, thedeformation energy of binding is taken to be the difference between theenergy of the free compound and the average energy of the conformationsobserved when the inhibitor binds to the enzyme.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include: Gaussian 94, revision C (Frisch,Gaussian, Inc., Pittsburgh, Pa. ©1995); AMBER, version 7. (Kollman,University of California at San Francisco, ©2002); QUANTA/CHARMM(Accelrys, Inc., San Diego, Calif., ©1995); Insight II/Discover(Accelrys, Inc., San Diego, Calif., ©1995); DelPhi (Accelrys, Inc., SanDiego, Calif., ©1995); and AMSOL (University of Minnesota) (QuantumChemistry Program Exchange, Indiana University). These programs may beimplemented, for instance, using a computer workstation, as are wellknown in the art, for example, a LINUX, SGI or Sun workstation. Otherhardware systems and software packages will be known to those skilled inthe art.

Those of ordinary skill in the art may express kinase protein usingmethods known in the art, and the methods disclosed herein. The nativeand mutated kinase polypeptides described herein may be chemicallysynthesized in whole or part using techniques that are well known in theart (see, e.g., Creighton, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., NY, 1983).

Gene expression systems may be used for the synthesis of native andmutated polypeptides. Expression vectors containing the native ormutated polypeptide coding sequence and appropriatetranscriptional/translational control signals, that are known to thoseskilled in the art may be constructed. These methods include in vitrorecombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, NY, 2001, and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and WileyInterscience, NY, 1989.

Host-expression vector systems may be used to express kinase. Theseinclude, but are not limited to, microorganisms such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing the coding sequence; yeast transformedwith recombinant yeast expression vectors containing the codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the coding sequence; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence; or animal cell systems. Theprotein may also be expressed in human gene therapy systems, including,for example, expressing the protein to augment the amount of the proteinin an individual, or to express an engineered therapeutic protein. Theexpression elements of these systems vary in their strength andspecificities.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector may contain: an origin of replication forautonomous replication in host cells, one or more selectable markers, alimited number of useful restriction enzyme sites, a potential for highcopy number, and active promoters. A promoter is defined as a DNAsequence that directs RNA polymerase to bind to DNA and initiate RNAsynthesis. A strong promoter is one that causes mRNAs to be initiated athigh frequency.

The expression vector may also comprise various elements that affecttranscription and translation, including, for example, constitutive andinducible promoters. These elements are often host and/or vectordependent. For example, when cloning in bacterial systems, induciblepromoters such as the T7 promoter, pL of bacteriophage λ, plac, ptrp,ptac (ptrp-lac hybrid promoter) and the like may be used; when cloningin insect cell systems, promoters such as the baculovirus polyhedrinpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (e.g., heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll a/b binding protein) or from plant viruses (e.g., the 35SRNA promoter of CaMV; the coat protein promoter of TMV) may be used;when cloning in mammalian cell systems, mammalian promoters (e.g.,metallothionein promoter) or mammalian viral promoters, (e.g.,adenovirus late promoter; vaccinia virus 7.5K promoter; SV40 promoter;bovine papilloma virus promoter; and Epstein-Barr virus promoter) may beused.

Various methods may be used to introduce the vector into host cells, forexample, transformation, transfection, infection, protoplast fusion, andelectroporation. The expression vector-containing cells are clonallypropagated and individually analyzed to determine whether they producethe appropriate polypeptides. Various selection methods, including, forexample, antibiotic resistance, may be used to identify host cells thathave been transformed. Identification of polypeptide expressing hostcell clones may be done by several means, including but not limited toimmunological reactivity with anti-kinase antibodies, and the presenceof host cell-associated activity.

Expression of cDNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell-basedsystems, including, but not limited, to microinjection into frogoocytes.

To determine the cDNA sequence(s) that yields optimal levels of activityand/or protein, modified cDNA molecules are constructed. A non-limitingexample of a modified cDNA is where the codon usage in the cDNA has beenoptimized for the host cell in which the cDNA will be expressed. Hostcells are transformed with the cDNA molecules and the levels of kinaseRNA and/or protein are measured.

Levels of kinase protein in host cells are quantitated by a variety ofmethods such as immunoaffinity and/or ligand affinity techniques,kinase-specific affinity beads or specific antibodies are used toisolate ³⁵S-methionine labeled or unlabeled protein. Labeled orunlabeled protein is analyzed by SDS-PAGE. Unlabeled protein is detectedby Western blotting, ELISA or RIA employing specific antibodies.

Following expression of kinase in a recombinant host cell, polypeptidesmay be recovered to provide the protein in active form. Severalpurification procedures are available and suitable for use. Recombinantkinase may be purified from cell lysates or from conditioned culturemedia, by various combinations of, or individual application of,fractionation, or chromatography steps that are known in the art.

In addition, recombinant kinase can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full length nascent protein orpolypeptide fragments thereof. Other affinity based purificationtechniques known in the art may also be used.

Alternatively, the polypeptides may be recovered from a host cell in anunfolded, inactive form, e.g., from inclusion bodies of bacteria.Proteins recovered in this form may be solubilized using a denaturant,e.g., guanidinium hydrochloride, and then refolded into an active formusing methods known to those skilled in the art, such as dialysis.

B. Cell Growth Assays

A variety of cell growth assays are known in the art and are useful inidentifying fused ring heterocycle compounds (i.e. “test compounds”)capable of inhibiting (e.g. reducing) cell growth and/or proliferation.

For example, a variety of cells are known to require specific kinasesfor growth and/or proliferation. The ability of such a cell to grow inthe presence of a test compound may be assessed and compared to thegrowth in the absence of the test compound thereby identifying theanti-proliferative properties of the test compound. One common method ofthis type is to measure the degree of incorporation of label, such astritiated thymidine, into the DNA of dividing cells. Alternatively,inhibition of cell proliferation may be assayed by determining the totalmetabolic activity of cells with a surrogate marker that correlates withcell number. Cells may be treated with a metabolic indicator in thepresence and absence of the test compound. Viable cells metabolize themetabolic indicator thereby forming a detectable metabolic product.Where detectable metabolic product levels are decreased in the presenceof the test compound relative to the absence of the test compound,inhibition of cell growth and/or proliferation is indicated. Exemplarymetabolic indicators include, for example tetrazolium salts andAlamorBlue® (see Examples section below).

V. Pharmaceutical Compositions and Administration

In another aspect, the present invention provides a pharmaceuticalcomposition including a fused ring heterocycle kinase modulator inadmixture with a pharmaceutically acceptable excipient. One of skill inthe art will recognize that the pharmaceutical compositions include thepharmaceutically acceptable salts of the fused ring heterocycle kinasemodulators described above.

In therapeutic and/or diagnostic applications, the compounds of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

The compounds according to the invention are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. A mostpreferable dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, camsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Preferred pharmaceuticallyacceptable salts include, for example, acetate, benzoate, bromide,carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate,mesylate, napsylate, pamoate (embonate), phosphate, salicylate,succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the invention may be formulated and dilutedin aqueous solutions, such as in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated.

For nasal or inhalation delivery, the agents of the invention may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with the inhibitors of this invention. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the inhibitors of this invention to treat proliferativediseases and cancer. Examples of known chemotherapeutic agents include,but are not limited to, adriamycin, dexamethasone, vincristine,cyclophosphamide, fluorouracil, topotecan, taxol, interferons, andplatinum derivatives.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation, anti-inflammatory agents suchas corticosteroids, TNF blockers, IL-1 RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; agents for treating diabetessuch as insulin, insulin analogues, alpha glucosidase inhibitors,biguanides, and insulin sensitizers; and agents for treatingimmunodeficiency disorders such as gamma globulin.

These additional agents may be administered separately, as part of amultiple dosage regimen, from the inhibitor-containing composition.Alternatively, these agents may be part of a single dosage form, mixedtogether with the inhibitor in a single composition.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those havingskill in the art from the foregoing description. Such modifications areintended to fall within the scope of the invention. Moreover, any one ormore features of any embodiment of the invention may be combined withany one or more other features of any other embodiment of the invention,without departing from the scope of the invention. For example, thefused ring heterocycle kinase modulators described in the Fused ringheterocycle kinase modulators section are equally applicable to themethods of treatment and methods of inhibiting kinases described herein.References cited throughout this application are examples of the levelof skill in the art and are hereby incorporated by reference herein intheir entirety for all purposes, whether previously specificallyincorporated or not.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. The preparation of embodiments of the presentinvention is described in the following examples. Those of ordinaryskill in the art will understand that the chemical reactions andsynthesis methods provided may be modified to prepare many of the othercompounds of the present invention. Where compounds of the presentinvention have not been exemplified, those of ordinary skill in the artwill recognize that these compounds may be prepared by modifyingsynthesis methods presented herein, and by using synthesis methods knownin the art.

Synthesis of the Compounds:

Method 1:

Step 1: Synthesis of 5-bromo-2-fluoro-pyridine-3-carbaldehyde

A solution of lithium di-iso-propylamine (5 mL, 35 mmol) in anhydrousTHF (40 mL) was cooled to −78° C. under nitrogen and n-butyl lithium(2.5 M in hexanes, 12 mL, 30 mmol) was added. The mixture was thenstirred at −78° C. for 15 min before 5-bromo-2-fluoro-pyridine (5 g, 28mmol) was added. The resulting mixture was then stirred at −78° C. for90 min. N-formylpiperidine (4 mL, 36 mmol) was added very rapidly to thesuspension at −78° C. and the mixture stirred vigorously for 60 sec. Thereaction was immediately quenched by the addition of a 10% (w/v) aqueoussolution of citric acid. The mixture was warmed to room temperature anddistributed between water and dichloromethane. The aqueous phase wasextracted three times with dichloromethane and the organic phases werecombined, dried over sodium sulfate, filtered and concentrated.Crystallization of the crude product from cyclohexane afforded5-bromo-2-fluoro-pyridine-3-carbaldehyde (2.993 g, 52% yield) as palebeige flaky crystals. ¹H-NMR (500 MHz, d₆-DMSO) δ 10.07 (s, 1H), 8.70(dd, 1H), 8.55 (dd, 1H). MS: m/z 236, 238 [MNa⁺], 204, 206 [MH⁺], 176,178 [MH-CO⁺].

Steps 2 and 3: Synthesis of 5-bromo-1H-pyrazolo[3,4-b]pyridine

5-bromo-2-fluoro-pyridine-3-carbaldehyde (13.66 g, 66.96 mmol), pinacol(8.75 g, 74.0 mmol) and para-toluenesulfonic acid monohydrate (1.50 g,7.89 mmol) were placed in a flask equipped with a DEAN-STARK-condenserand dissolved in anhydrous benzene (400 mL). The mixture was heated toreflux and solvent distilled off until the distillate remains clear andthe remaining volume was approximately 200 ml. The mixture was dilutedwith ethyl acetate (300 mL) and washed with a saturated aqueous solutionof sodium bicarbonate and brine, then dried over sodium sulfate,filtered and concentrated. The resulting residue was dissolved in amixture of ethanol (400 mL) and di-iso-propyl-ethyl-amine (25 mL).Anhydrous hydrazine (15 ml, 0.48 mol) was then added and the resultingmixture was stirred under reflux conditions for 4 h. The mixture wasthen concentrated to dryness and the resulting residue was distributedbetween water and toluene. The organic phase was washed with brinetwice, dried over sodium sulfate, filtered and concentrated. The residuewas dissolved in anhydrous ether (700 mL) and hydrogen chloride inanhydrous ether (2M, 70 mL) was added slowly to the vigorously stirredsolution. The precipitate was filtered off, washed with ether and hexaneand then dried in vacuum. ¹H-NMR (500 MHz, d₆-DMSO) δ 10.31 (s, br, 1H),8.86 (s, 1H), 8.37 (d, 1H), 7.88 (d, 1H), 6.08 (s, 1H), 3.56 (s, br),1.27 (s, 6H), 1.19 (s, 6H). MS: m/z 198, 200 [MH⁺].

The above solid was dissolved in a mixture of water (500 mL), ethanol(200 mL) and concentrated aqueous hydrochloric acid (50 mL) at 50-65° C.The mixture was then stirred at room temperature for 16 h before beingneutralized to pH=8 with sodium bicarbonate. The resulting precipitatewas filtered off and the aqueous phase extracted three times with ethylacetate. The combined organic phases are washed with brine, dried oversodium sulfate, filtered and concentrated. The resulting residue and theprecipitate obtained are crystallized from ethanol to afford5-bromo-1H-pyrazolo[3,4-b]pyridine (6.615 g, 50% yield) as a crystallinebeige to pale olive-green solid. ¹H-NMR (500 MHz, d₆-DMSO) δ 13.91 (s,1H), 8.60 (d, 1H), 8.54 (d, 1H), 8.16 (s, br, 1H). MS: m/z 198, 200[MH⁺].

Step 4: Synthesis of 5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine

5-bromo-1H-pyrazolo[3,4-b]pyridine (3.00 g, 15.2 mmol) andN-iodosuccinimide (3.60 g, 16.0 mmol) were dissolved in anhydrousdichloroethane (100 mL). The resulting mixture was stirred under refluxconditions for 6 h, cooled to room temperature and diluted with THF (300mL). The resulting solution was washed with a saturated aqueous solutionof sodium thiosulfate (100 mL) and brine, then dried over magnesiumsulfate, filtered and concentrated. The residue was titurated with a 1:1mixture of dichloromethane and ether and then ether before being driedin vacuum to afford 5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (3.795 g,77% yield) as a beige-brown solid. ¹H-NMR (500 MHz, d₆-DMSO) δ 14.31 (s,1H), 8.65 (d, 1H), 8.20 (d, 1H). MS: m/z 323, 325 [MH⁺].

Step 5: Synthesis of5-bromo-3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine

Under nitrogen 5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (2.68 g, 8.27mmol) was dissolved in anhydrous DMF (40 mL). The solution was cooled to0-5° C. and an excess of dry sodium hydride added until further additiondoes not result in hydrogen formation. To the resulting suspension wasadded 2-trimethylsilanyl-ethoxymethylchloride (2.5 ml, 14 mmol) dropwise at 0-5° C. The resulting mixture was stirred at 0° C. for 1 h andthereafter quenched by addition of methanol and subsequently of asaturated aqueous solution of ammonium chloride. The mixture was thenconcentrated to dryness at 50° C. under reduced pressure. The resultingresidue was distributed between water, brine and dichloromethane. Theaqueous phase was then extracted with dichloromethane and the combinedorganic phases were dried over sodium sulfate, filtered andconcentrated. The crude product was purified by flash silica gelchromatography using a gradient of ethyl acetate in hexanes to afford5-bromo-3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(2.929 g, 78% yield) as a beige to brown solid. ¹H-NMR (500 MHz,d₆-DMSO) δ 8.85 (d, 1H), 8.40 (d, 1H), 5.85 (s, 2H), 3.69 (t, 2H), 0.92(t, 2H), 0.11 (s, 9H).

Step 6: Synthesis of5-bromo-3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine

A mixture of5-bromo-3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(1.606 g, 3.537 mmol), 2-methoxy-phenyl-boronic acid (575 mg, 3.78 mmol)and of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (145 mg, 0.178 mmol) in acetonitrile (8 mL) andaqueous solution of sodium carbonate (2M, 8 mL) was stirred in a closedvial at 85° C. for 100 min. The resulting mixture was then distributedbetween a saturated aqueous solution of sodium bicarbonate anddichloromethane and the aqueous phase extracted three times withdichloromethane. The combined organic phases were dried over sodiumsulfate, filtered and concentrated. The crude product was purified byflash silica gel chromatography using a gradient of ethyl acetate inhexanes to afford5-bromo-3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(1.002 g, 65% yield) as an off-white oil. ¹H-NMR (500 MHz, d₆-DMSO) δ8.70 (d, 1H), 8.40 (d, 1H), 7.61 (d, 1H), 7.50 (ddd, 1H), 7.23 (dd, 1H),7.10 (ddd, 1H), 5.81 (s, 2H), 3.85 (s, 3H), 3.66 (t, 2H), 0.84 (t, 2H),−0.10 (s, 9H). MS: m/z 456, 458 [MNa⁺].

Step 7: Synthesis of3-(2-methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine

Bis(pinacolato)diboron (1.20 g, 4.73 mmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (100 mg, 0.122 mmol) and anhydrous sodium acetate(625 mg, 7.62 mmol) were placed in a nitrogen flushed vial. To this wasadded a solution of5-bromo-3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(1.002 g, 2.307 mmol) in anhydrous DMF (15 mL). The resulting mixturewas irradiated in a Personal Chemistry Optimizer at 130° C. for 60 minand then concentrated at 50° C. under reduced pressure. The resultingresidue was distributed between ether and brine and the aqueous phasewas extracted with ether. The organic phases were combined, dried oversodium sulfate, filtered and concentrated. The crude product was thenpurified by flash silica gel chromatography using a gradient of ethylacetate in hexanes to afford3-(2-methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(1.370 g, 123% yield) as a pale olive-green solid. ¹H-NMR (500 MHz,d₆-DMSO) 68.76 (d, 1H), 8.40 (d, 1H), 7.59 (dd, 1H), 7.51 (ddd, 1H),7.25 (m, 1H), 7.12 (ddd, 1H), 5.84 (s, 2H), 3.82 (s, 3H), 3.67 (t, 2H),1.33 (s, 12H), 0.84 (t, 2H), −0.10 (s, 9H).

Step 8: Synthesis of{2-hydroxy-5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone

A mixture of3-(2-methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(100 mg, 0.21 mmol), (5-bromo-2-hydroxy-phenyl)-morpholin-4-yl-methanone(66 mg (0.23 mmol) and1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (9 mg, 11 μmol) in acetonitrile (2 mL) and aqueoussolution of sodium carbonate (2M, 2 mL) was irradiated in a PersonalChemistry Optimizer at 135° C. for 20 min. The crude reaction mixturewas distributed between dichloromethane and a saturated aqueous solutionof sodium bicarbonate. The aqueous phase was then extracted withdichloromethane and the combined organic phases were dried over sodiumsulfate, filtered and concentrated. The crude product was then purifiedby flash silica gel chromatography using a gradient of ethyl acetate inhexanes to afford{2-hydroxy-5-[3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(35 mg, 30% yield) as a colorless solid. ¹H-NMR (500 MHz, d₆-DMSO) δ8.86 (d, 1H), 8.27 (d, 1H), 7.64 (dd, 1H), 7.62 (dd, 1H), 7.54 (d, 1H),7.49 (ddd, 1H), 7.24 (d, br, 1H), 7.11 (ddd, 1H), 7.00 (d, 1H), 5.84 (s,2H), 3.84 (s, 3H), 3.69 (t, 2H), 3.7-3.2 (m, 8H), 0.86 (t, 2H), −0.08(s, 9H). MS: m/z 583 [MNa⁺], 561 [MH⁺], 443 [MH⁺-(Me₃Si(CH₂)₂O)]

A solution of{2-hydroxy-5-[3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(34 mg, 61 μmol) in dichloromethane (15 mL) was cooled to 0-5° C. andboron trifluoride diethyl etherate (100 μl, 0.8 mmol) was added. Themixture was then stirred at 0-5° C. for 40 min before 10 ml of a 10%(w/v) solution of potassium hydroxide was added. The mixture was furtherstirred at room temperature for 1 h. The pH was then adjusted toapproximately 3-4 by addition of citric acid and the aqueous phasesaturated with sodium sulfate. The resulting mixture was extracteddichloromethane (3×). The organic phases were combined, washed with asaturated aqueous solution of sodium bicarbonate, dried over sodiumsulfate and evaporated to afford{2-hydroxy-5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(11.5 mg, 44% yield) as a colorless solid. ¹H-NMR (500 MHz, d₆-DMSO) δ13.76 (s, 1H), 10.06 (s, 1H), 8.78 (d, 1H), 8.23 (d, 1H), 7.64 (dd, 1H),7.62 (dd, 1H), 7.51 (d, 1H), 7.46 (ddd, 1H), 7.22 (d, 1H), 7.10 (t, 1H),6.99 (d, 1H), 3.84 (s, 3H), 3.7-3.2 (m, 8H). MS: m/z 431 [MH⁺].

Other Compounds Prepared by Method 1:

TABLE 1 Structure

MS: m/z 375 (M + H⁺)Method 2:

Step 1: Synthesis ofmorpholin-4-yl-[3-(1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-methanone

A mixture of 5-bromo-1H-pyrazolo[3,4-b]pyridine (1.50 g, 7.57 mmol),3-(morpholin-4-carbonyl)phenylboronic acid (2.136 g, 9.09 mmol) andtetrakis(triphenylphosphine)palladium(0) (435 mL, 0.376 mmol) indimethoxyethane (8 mL) and saturated aqueous solution of sodiumbicarbonate (8 mL) was irradiated in a Personal Chemistry Optimizer at175° C. for 60 min. The crude reaction mixture was distributed betweendichloromethane and a saturated aqueous solution of sodium bicarbonate.The aqueous phase was then extracted with dichloromethane, and thenethyl acetate and the combined organic phases were dried over sodiumsulfate, filtered and concentrated to afford a pale green foamcontaining 80% ofmorpholin-4-yl-[3-(1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-methanone(2.30 g, 80% yield) and 20% of triphenylphosphine oxide. ¹H-NMR (500MHz, d₆-DMSO) δ 13.75 (s, 1H), 8.87 (d, 1H), 8.54 (d, 1H), 8.21 (d, 1H),7.85 (m, 1H), 7.77 (m, 1H), 7.58 (t, 1H), 7.41 (m, 1H).

Step 2: Synthesis of[3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-morpholin-4-yl-methanone

Morpholin-4-yl-[3-(1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-methanone(2.30 g, 80% pure, ˜6 mmol) and of N-iodosuccinimide (2.50 g, 11.1 mmol)were dissolved in dichloroethane (180 mL). The mixture was stirred underreflux conditions for 5 h, then cooled to room temperature and dilutedwith dichloromethane. The solution was washed with saturated aqueoussolution of sodium thiosulfate (1×) and then with a saturated aqueoussolution of sodium bromide (2×), dried over sodium sulfate, filtered andconcentrated. The resulting residue was washed with ether (80 mL) anddried to afford[3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-morpholin-4-yl-methanoneas a beige powder (2.881 g, 88% yield over two steps). ¹H-NMR (500 MHz,d₆-DMSO) δ 14.19 (s, 1H), 8.92 (d, 1H), 8.14 (d, 1H), 7.91 (m, 1H), 7.83(m, 1H), 7.59 (ddd, 1H), 7.44 (dt, 1H), 3.75-3.35 (m, 8H).

Step 3: Synthesis ofmorpholin-4-yl-{3-[3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-methanone

A mixture of[3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-morpholin-4-yl-methanone(25 mg, 58 μmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (5 mg, 6 μmol) and 1H-pyrazol-4-ylboronic acid (11mg, 98 μmol) in acetonitrile (2 mL) and 2 M solution of sodium carbonate(1 mL) was irradiated in a Personal Chemistry Optimizer at 175° C. for30 min. The crude reaction mixture was diluted with water (1 mL) andethyl acetate (3 mL) and the organic phase separated, filtered andconcentrated. The resulting crude mixture was then purified bymass-triggered reverse phase HPLC using a gradient of acetonitrile inwater containing 0.1% of formic acid to affordmorpholin-4-yl-{3-[3-(1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-methanone(6.2 mg, 29% yield) of as a colorless powder. ¹H-NMR (500 MHz, d₆-DMSO)δ 13.59 (s, 1H), 13.17 (s, 1H), 8.87 (d, 1H), 8.73 (d, 1H), 8.60 (s, br,1H), 8.17 (s, br, 1H), 7.95 (ddd, 1H), 7.89 (t, 1H), (t, 1H), 7.59 (t,1H), 7.43 (ddd, 1H), 3.80-3.35 (m, 8H). MS: m/z 397 [MNa⁺], 375 [MH⁺].

Other compounds prepared by Method 2:

TABLE 2 Structure

MS: m/z 415 (M + H⁺)

MS: m/z 417 (M + H⁺)

MS: m/z 424 (M + H⁺

MS: m/z 413 (M + H⁺

MS: m/z 433 (M + H⁺)

MS: m/z 447 (M + H⁺)

MS: m/z 429 (M + H⁺)

MS: m/z 405 (M + H⁺)

MS: m/z 429 (M + H⁺)Method 3:

Step 1: Synthesis of{3-[3-Iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone

To a solution of[3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)-phenyl]-morpholin-4-yl-methanone(2.12 g, 4.88 mmol) in anhydrous DMF (30 mL) was added sodium hydride(60% in mineral oil, 750 mg, 30 mmol) at 0-5° C. The mixture was stirredfor a few minutes before trimethylsilylethoxymethyl chloride (2.0 ml, 11mmol) was added drop wise at the same temperature. The mixture wasstirred at 0° C. to room temperature for 4 hours and then cooled to 0-5°C. and quenched by an addition of methanol. The resulting suspension wasthen distributed between water, saturated aqueous ammonium chloridesolution and ether. The aqueous phase was extracted three times withether and the combined organic phases were dried over sodium sulfate,filtered and concentrated. The crude product was then purified by silicagel chromatography using a gradient of ethyl acetate in hexanes toafford{3-[3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanoneas a beige-brown foam (1.806 g, 66% by ¹H-NMR, side product identifiedasmorpholin-4-yl-{3-[2-(2-trimethylsilanyl-ethoxymethyl)-2H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-methanone).¹H-NMR (500 MHz, d₆-DMSO) δ 9.08 (d, 1H), 8.29 (d, 1H), 8.01 (m, 1H),7.95 (t, br, 1H], 7.69 (t, 1H), 7.55 (d, br, 1H), 5.88 (s, 2H), 3.71 (t,2H), 3.85-3.45 (m, 8H), 0.94 (t, 2H), −0.2 (s, 9H). MS: m/z 565 [MNa⁺],537 [MH⁺], 447 [MH⁺-(Me₃ Si(CH₂)₂O)].

Step 2: Synthesis of{3-[3-(2-methoxy-pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone

A mixture of{3-[3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(33 mg, 66% pure, 38 μmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (5 mg, 6 μmol) and 3-trifluoromethylphenylboronicacid (14 mg, 92 μmol) in acetonitrile (2 mL) and 2 M solution of sodiumcarbonate (1 mL) was irradiated in a Personal Chemistry Optimizer at175° C. for 20 min. The crude reaction mixture was diluted withsaturated aqueous solution of sodium bromide (1 mL) and ethyl acetate (4mL) and the organic phase separated, adsorbed onto silica and purifiedby flash silica gel chromatography using a gradient of ethyl acetate inhexanes to afford{3-[3-(2-methoxy-pyridin-3-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(24 mg, 116% yield) as an off-white residue. MS: m/z 568 [MNa⁺], 546[MH⁺], 428 [MH⁺-(Me₃Si(CH₂)₂O)]

This residue was dissolved in THF (2 ml) and activated 4 Å molecularsieves were added to the mixture. Tetra-n-butylammonium fluoride in THF(1 M solution, 0.5 ml, 0.5 mmol) was added and the mixture stirred at70° C. for 26 h. The mixture was cooled to room temperature and 1 ml ofcation exchange resin (Amberlyst, Na⁺-form) added and the mixture wasshaken for 40 min. The resin and sieves were then filtered off, washingwith dichloromethane and methanol and the filtrate obtained wasconcentrated. The residue was dissolved in ethyl acetate and purified byflash chromatography on silica gel using a gradient of ethyl acetatecontaining 15% (v/v) of methanol in ethyl acetate. The product fractionswas combined, concentrated and purified by mass-triggered reverse phaseHPLC using a gradient of acetonitrile in water containing 0.1% of formicacid to afford{3-[3-(2-methoxy-pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone(3.2 mg, 20% yield) as a colorless solid. ¹H-NMR (500 MHz, d₆-DMSO) δ14.01 (s, 1H), 8.91 (d, 1H), 8.51 (d, 1H), 8.31 (dd, 1H], 8.11 (dd, 1H),7.87 (ddd, 1H), 7.80 (t, 1H), 7.59 (t, 1H), 7.43 (dt, 1H), 7.18 (dd,1H), 3.97 (s, 3H), 3.70-3.35 (m, 8H). MS: m/z 416 [MH⁺].

Other compounds prepared by Method 3:

TABLE 3 Structure

MS: m/z 453 (M + H⁺)

MS: m/z 420 (M + H⁺)

MS: m/z 421 (M + H⁺)

MS: m/z 469 (M + H⁺)

MS: m/z 438 (M + H⁺)

MS: m/z 436 (M + H⁺)Method 4:

Step 1: Synthesis ofN,N-dimethyl-3-(5-(3-(morpholine-4-carbonyl)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzamide

A mixture of(3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(morpholino)methanone(50 mg, 0.089 mmol), 3-(dimethylcarbamoyl)phenylboronic acid (34 mg,0.177 mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)complex with dichloromethane (3.6 mg, 0.0045 mmol) and sodium carbonate(2M aqueous solution, 0.134 mL, 0.267 mmol) in acetonitrile (1 mL) washeated in a Personal microwave at 90° C. for 30 min. The resultingmixture was diluted with water and extracted with ethyl acetate. Theorganic layers were combined, dried over sodium sulfate, filtered andconcentrated to dryness. Silica gel chromatography purification of thecrude product affordN,N-dimethyl-3-(5-(3-(morpholine-4-carbonyl)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzamideas a clear oil. MS: m/z 586.2 [M+H⁺].

Step 2: Synthesis ofN,N-dimethyl-3-(5-(3-(morpholine-4-carbonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzamide

A solution ofN,N-dimethyl-3-(5-(3-(morpholine-4-carbonyl)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzamidein 5% of perchloric acid in acetic acid (1 mL) was stirred for 1 h atroom temperature. Saturated sodium bicarbonate solution was then addedto the solution slowly until pH ˜8 and the mixture was stirred for 24hours at room temperature. Ethyl acetate was then used for extractionand the organic layers were combined, dried over sodium sulfate,filtered and concentrated to dryness. Mass triggered reverse phase HPLCpurification affordedN,N-dimethyl-3-(5-(3-(morpholine-4-carbonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzamide(7.4 mg, 18% yield over two steps) as light yellow solids. ¹H NMR (500MHz, CDCl₃) δ 2.99 (s, 3H), 3.10 (s, 3H), 3.46 (br, 2H), 3.61 (br, 2H),3.76 (br, 4H), 7.40 (d, J=7.0 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.52 (m,2H), 7.64 (d, J=7.5 Hz, 1H), 7.68 (s, 1H), 7.98 (m, 2H), 8.62 (s, 1H),8.85 (br, 1H). MS: m/z 456.1 (M+H⁺).

Other Compounds Prepared by Method 4:

The conditions in step 2 may vary for the compounds in the followingtable. Sometimes sodium carbonate may be needed instead of sodiumbicarbonate and the reaction time varies from 30 minutes to 24 hours.

TABLE 4 Structure

MS: m/z 405.1 (M + H⁺)

MS: m/z 449.0 (M + H⁺)

MS: m/z 417.1 (M + H⁺)

MS: m/z 403.1 (M + H⁺)

MS: m/z 433.1 (M + H⁺)

MS: m/z 416 (M + H⁺)

MS: m/z 421.1 (M + H⁺)

MS: m/z 419.1 (M + H⁺)

MS: m/z 449 (M + H⁺)

MS: m/z 410.1 (M + H⁺)

MS: m/z 399.1 (M + H⁺)

MS: m/z 433 (M + H⁺)

MS: m/z 447.1 (M + H⁺)

MS: m/z 443.1 (M + H⁺)

MS: m/z 473.1 (M + H⁺)

MS: m/z 437.0 (M + H⁺)

MS: m/z 415.1 (M + H⁺)

MS: m/z 427.1 (M + H⁺)

MS: m/z 447.1 (M + H⁺)

MS: m/z 428.0 (M + H⁺)

MS: m/z 433.1 (M + H⁺).

MS: m/z 453.0 (M + H⁺)Method 5:

Step 1: Synthesis of methyl 3-(1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

A mixture of 5-bromo-1H-pyrazolo[3,4-b]pyridine (2.00 g, 10.10 mmol),3-(methoxycarbonyl)phenylboronic acid (2.20 g, 12.12 mmol), sodiumbicarbonate (2.2 g, 6.00 mmol), andtetrakis(triphenylphosphine)palladium(0) (0.250 g, 0.202 mmol) indioxane/water (40 mL/10 mL) was stirred at 110° C. for 15 hours. Themixture was then poured into ice water and extracted with ethyl acetate(3×). The organic layers were combined, dried over sodium sulfate,filtered and concentrated to dryness. Silica gel chromatography of thecrude product afforded methyl 3-(1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate(8) (1.65 g, 65% yield) as yellow solids. MS: m/z 254.0 (M+H⁺).

Step 2: Synthesis of methyl3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate

To a solution of methyl 3-(1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (1.65g, 6.52 mmol) in dichloroethane (40 mL) was added NIS (1.81 g, 8.04mmol) and the mixture was stirred at 70° C. for 6 hours. The solvent wasremoved by reduced pressure and the crude product was purified by silicagel chromatography to afforded methyl3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (9) (988 mg, 40%yield). MS: m/z 379.9 (M+H⁺).

Step 3: Synthesis of3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoicacid

To a solution of methyl3-(3-iodo-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (988 mg, 2.61 mmol) inDMF was added sodium hydride (60% in mineral oil, 525 mg, 13.03 mmol) at−40° C. The mixture was stirred for 60 minutes before SEMCl (920 μl,5.22 mmol) was added. The reaction was warmed to room temperature andquenched with methanol and water. Acetic acid was then used to adjustthe pH to 4-5. The mixture was then extracted with ethyl acetate (3×)and the organic layers were combined, dried over sodium sulfate,filtered and concentrated to dryness. Silica gel chromatographypurification of the resulting crude product afforded3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoicacid (200 mg, 15% yield) as solids. MS: m/z 517.9 (M+Na⁺).

Step 4: Synthesis of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone

A mixture of crude3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoicacid (200 mg, 0.404 mmol), N,N-dimethyl-2-(piperazin-1-yl)ethanamine (76mg, 0.485 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (185 mg, 0.485 mmol), triethyl amine (0.700 ml,0.485 mmol) and in DMF (2 ml) was stirred at 90° C. in PersonalMicrowave for 1 hour. Water was then added to the mixture and extractedwith ethyl acetate (3×). The organic layers were combined, dried oversodium sulfate, filtered and concentrated to dryness. Silica gelchromatography of the crude product afforded(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone(110 mg, 43% yield) as off-white solids. MS: m/z 635.1 (M+H⁺).

Step 5: Synthesis of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-(5-fluoro-2-methoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone

A mixture of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone(50 mg, 0.079 mmol), 5-fluoro-2-methoxyphenylboronic acid (20 mg, 0.118mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)complex with dichloromethane (7.9 mg, 0.10 mmol) and sodium carbonate(2M aqueous solution, 0.119 mL, 0.237 mmol) in acetonitrile (1 mL) washeated in a Personal microwave at 90° C. for 30 min. The resultingmixture was diluted with water and extracted with ethyl acetate. Theorganic layers were combined, dried over sodium sulfate, filtered andconcentrated to dryness. Silica gel chromatography purification of thecrude product afford(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-(5-fluoro-2-methoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanoneas a light yellow oil. MS: m/z 633.3 (M+H⁺).

Step 6: Synthesis of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-(5-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone

A solution of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-(5-fluoro-2-methoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanonefrom Step 5 in 5% of perchloric acid in methanol (1 mL) was stirred for45 minutes at room temperature. Sodium hydroxide solution (2M) was thenadded to the solution slowly until pH ˜8. Ethyl acetate was then usedfor extraction and the organic layers were combined and concentrated todryness, which was then redissolved in methanol (1 mL) and sodiumcarbonate (2M, 1 mL). The mixture was stirred at room temperature for 15hours before being diluted with water and extracted with ethyl acetate(3×). The organic layers were combined, dried over sodium sulfate,filtered and concentrated to dryness. Mass triggered reverse phase HPLCpurification afforded(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-(5-fluoro-2-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone(25.6 mg, 64% yield from 12) as white solids. ¹H NMR (500 MHz, CD₃O)) δ2.42 (s, 6H), 2.49 (br, 2H), 2.59 (m, 4H), 2.69 (m, 2H), 3.54 (br, 2H),3.81 (br, 2H), 3.85 (s, 3H), 7.17 (m, 2H), 7.40 (dd, J=3.0, 9.5 Hz, 1H),7.44 (d, br, J=7.5 Hz, 1H), 7.59 (t, J=7.5 Hz, 1H), 7.72 (br, 1H), 7.77(d, J=8.5 Hz, 1H), 8.39 (d, J=1.5 Hz, 1H), 8.80 (d, J=2.5 Hz, 1H). MS:m/z 503.2 (M+H⁺).

Method 6:

Step 1: Synthesis of(3-(3-(2,6-dimethoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(4-(2-(dimethylamino)ethyl)piperazin-1-yl)methanone

A mixture of(4-(2-(dimethylamino)ethyl)piperazin-1-yl)(3-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)methanone(50 mg, 0.079 mmol), 2,6-dimethoxyphenylboronic acid (22 mg, 0.118mmol), tetrakis(triphenylphosphine)palladium(0) (9.1 mg, 0.10 mmol) andsodium carbonate (2M aqueous solution, 0.119 mL, 0.237 mmol) inacetonitrile (1 mL) was heated in a Personal microwave at 120° C. for 30min. The resulting mixture was diluted with water and extracted withethyl acetate. The organic layers were combined, dried over sodiumsulfate, filtered and concentrated to dryness. Silica gel chromatographypurification of the crude product afford(3-(3-(2,6-dimethoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(4-(2-(dimethylamino)ethyl)piperazin-1-yl)methanoneas a clear oil. MS: m/z 645.3 (M+H⁺).

Step 2: Synthesis of(3-(3-(2,6-dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(4-(2-(dimethylamino)ethyl)piperazin-1-yl)methanone

A solution of(3-(3-(2,6-dimethoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(4-(2-(dimethylamino)ethyl)piperazin-1-yl)methanonein 5% of perchloric acid in acetic acid (1 mL) was stirred for 45minutes at room temperature. Sodium hydroxide solution (2M) was thenadded to the solution slowly until pH ˜8. Ethyl acetate was then usedfor extraction and the organic layers were combined and concentrated todryness, which was then redissolved in methanol (1 mL) and sodiumcarbonate (2M, 1 mL). The mixture was stirred at room temperature for 15hours before being diluted with water and extracted with ethyl acetate(3×). The organic layers were combined, dried over sodium sulfate,filtered and concentrated to dryness. Mass triggered reverse phase HPLCpurification afforded(3-(3-(2,6-dimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)phenyl)(4-(2-(dimethylamino)ethyl)piperazin-1-yl)methanone(22.70 mg, 56% yield for two steps) as white solids. ¹H NMR (500 MHz,CD₃OD) δ 2.48 (br, 2H), 2.52 (s, 6H), 2.60 (m, 4H), 2.69 (m, 2H), 3.51(br, 2H), 3.74 (s, 6H), 3.80 (br, 2H), 6.80 (d, J=8.5 Hz, 2H), 7.43 (m,2H), 7.56 (t, J=8.0 Hz, 1H), 7.69 (s, 1H), 7.75 (d, J=8.0 Hz, 1H), 8.09(d, J=2 Hz, 1H), 8.81 (d, J=2 Hz, 1H). MS: m/z 515.2 (M+H⁺).

Other Compounds Prepared by Method 6:

The conditions in step 2 may vary for the compounds in the followingtable. Sometimes sodium carbonate may be needed instead of sodiumbicarbonate and the reaction time varies from 30 minutes to 24 hours.

TABLE 5 Structure

MS: m/z 449.1 (M + H⁺)

MS: m/z 433.1 (M + H⁺)

MS: m/z 413.1 (M + H⁺)

MS: m/z 447.1 (M + H⁺)

MS: m/z 453.0 (M + H⁺) * Step 1 Suzuki coupling conditions: 150° C., 1h, μw. ** Step 1 Suzuki coupling conditions: 120° C., 1 h, μw. *** Step1 Suzuki coupling conditions: Pd₂(dba)₃, K₃PO₄, anddicyclohexylphenylphosphine, CH₃CN, 150° C., 4 h, μw.Method 7:

Step 1: Synthesis of5-[3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinicacid ethyl ester

A mixture of 3-ethoxycarbonyl-5-pyridylboronic acid (529 mg, 1.91 mmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (66 mg, 0.09 mmol) and5-bromo-3-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(780 mg, 1.80 mmol) acetonitrile (5 mL) and 2 M aqueous solution ofsodium carbonate (5 mL) were added and the mixture was irradiated in aPersonal Chemistry Optimizer to 90° C. for 30 minutes. The crudereaction mixture was distributed between ethyl acetate and water. Theaqueous phase was extracted three times with ethyl acetate and thecombined organic phases were dried over sodium sulfate, filtered andconcentrated. The crude product was purified by flash silica gelchromatography using a gradient of ethyl acetate in hexanes to afford5-[3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinicacid ethyl ester (552 mg, 61% yield) as a yellow oil. ¹H-NMR (500 MHz,d₆-DMSO) δ 9.24 (d, 1H), 9.12 (d, 1H), 9.03 (d, 1H), 8.60 (t, 1H), 8.56(d, 1H), 7.66 (dd, 1H), 7.51 (ddd, 1H), 7.25 (dd, 1H), 7.12 (dt, 1H),5.88 (s, 2H), 4.40 (q, 2H), 3.87 (s, 3H), 3.71 (t, 2H), 1.37 (t, 3H),0.87 (t, 2H), −0.073 (t, 9H). MS: m/z 505 [MH⁺].

Step 2: Synthesis of5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinic acid

To a solution of5-[3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinicacid ethyl ester (494 mg, 0.98 mmol) in THF (20 mL) was addedtetra-n-butylammonium fluoride in THF (1M, 10 ml, 10 mmol) and activated4 Å molecular sieves. The resulting mixture was stirred at 70° C. for 7h. The sieves were filtered off, washing with ethyl acetate and theresulting filtrate concentrated. The residue was distributed betweendichloromethane and water. The aqueous phase was extracted three timeswith dichloromethane and the organic phases combined, dried over sodiumsulfate, filtered and concentrated to afford of5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinic acid(654 mg, 62% purity, 405 mg, 119% yield) as a brown solid. ¹H-NMR (500MHz, d₆-DMSO) δ 13.98 (s, 1H), 8.99 (d, 1H), 8.95 (s, 1H), 8.88 (d, 1H),8.43 (t, 1H), 8.40 (d, 1H), 7.67 (dd, 1H), 7.47 (ddd, 1H), 7.23 (d, 1H),7.10 (dt, 1H), 3.86 (s, 3H). MS: m/z 345 (96%) [M−H⁻].

Step 3: Synthesis of{5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-pyridin-3-yl}-pyrrolidin-1-yl-methanone

(5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinic acid(350 mg, 62% pure, 0.63 mmol) was dissolved in anhydrous DMF (20 mL) at50-60° C. and the solution was cooled to room temperature when PS-HOBtresin (Argonaut Technologies) (0.9 mmol·g⁻¹ loading, 2.20 g, 1.98 mmol),DMAP (32 mg, 0.26 mmol) and EDCI (375 mg, 1.95 mmol) were added. Themixture was shaken at room temperature for 16 h. The resin was filteredoff, washing six times with DMF and subsequently three times with etherand dried. The resin and(5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-nicotinate)(460 mg, theoretical loading 105 μmol) were suspended in anhydrous DMF(3 mL) containing pyrrolidine (110 μl, 1.3 mmol) and shaken for 22 h.The resin is filtered off, washing with dichloromethane, ether and DMF.The filtrate and washings were combined and concentrated. The resultingresidue is purified by mass-triggered reverse phase HPLC using agradient of acetonitrile in water containing 0.1% of formic acid toafford{5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-pyridin-3-yl}-pyrrolidin-1-yl-methanone(2.4 mg, 6 μmol, 6% yield) as a light brown solid. ¹H-NMR (500 MHz,d₆-MeOH) δ 68.98 (d, 1H), 8.87 (d, 1H), 8.74 (d, 1H), 8.50 (d, 1H), 8.31(t, 1H), 7.67 (dd, 1H), 7.48 (ddd, 1H), 7.21 (d, 1H), 7.11 (dt, 1H),3.89 (s, 3H), 3.65 (t, 2H), 3.58 (t, 2H), 2.02 (m, 2H), 1.96 (m, 2H).MS: m/z 400 [MH⁺].

Other compounds prepared by Method 7:

TABLE 6 Structure

MS: m/z 417 [MH⁺]

MS: m/z 402 [MH⁺]

MS: m/z 443 [MH⁺]

MS: m/z 386 [MH⁺]

MS: m/z 374 [MH⁺]

MS: m/z 414 [MH⁺]

MS: m/z 486 [MH⁺]

MS: m/z 473 [MH⁺]

MS: m/z 526 [MH⁺]

MS: m/z 542 [MH⁺]

MS: m/z 385 [MH⁺]

MS: m/z 416 [MH⁺]

MS: m/z 472 [MH⁺]

MS: m/z 525 [MH⁺]

MS: m/z 541 [MH⁺]

MS: m/z 399 [MH⁺]

MS: m/z 413 [MH⁺]

MS: m/z 442 [MH⁺]

MS: m/z 443 [MH⁺]

MS: m/z 379 [MH+]

MS: m/z 421 [MH+]

MS: m/z 405 [MH+] * synthesized starting from3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid andpurified by flash chromatography on silica gel using a gradient of 20%v/v methanol in ethyl acetate in hexanes ** synthesized starting from3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acidMethod 8:

Step 1: Synthesis of[4-(2-dimethylamino-ethyl)-piperazin-1-yl]-{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-methanone

3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid(338 mg, 0.79 mmol) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (300 mg, 0.79 mmol) were dissolved in a mixture of20 ml of acetonitrile and 10 ml of methanol. 131 mg (0.83 mmol) of1-(2-dimethylaminoethyl)-piperazine was added and the mixture stirred atambient temperature for 6 h. The resulting mixture was distributedbetween dichloromethane and a 2 M aqueous solution of sodium carbonate.The phases were separated and the aqueous layer extracted three timeswith dichloromethane. The combined organic layers were combined, washedwith a saturated aqueous solution of sodium bromide, dried over sodiumsulfate, and evaporated. The crude material was purified by flashchromatography on silica gel using a stepped gradient of ethyl acetateand a 4:4:1 solvent mixture of ethyl acetate, dichloromethane andmethanol containing 2% v/v of 35% wt ammonia in water to afford[4-(2-dimethylamino-ethyl)-piperazin-1-yl]-{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-methanone(160 mg, 42%) as an oil. ¹H-NMR (d₆-CDCl₃) δ 8.87 (d) [1H], 8.36 (d)[1H], 7.75 (dd) [1H], 7.68 (t) [1H], 7.67 (m) [1H], 7.53 (m) [1H], 7.46(mt) [1H], 7.42 (md) [1H], 7.13 (dt) [1H], 7.10 (d) [1H], 3.89 (s) [3H],3.81-3.86 (m) [2H], 3.48-3.55 (m) [2H], 2.58-2.64 (m) [2H], 2.56 (m)[2H], 2.50 (m) [2H], 2.44-2.52 (m) [2H]. MS: m/z 485 (M+H⁺).

Method 9:

Step 1: Synthesis of5-bromo-3-iodo-1-(2-methoxy-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine

To a solution of 5-bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (470 mg, 1.45mmol), 60% sodium hydride in mineral oil (104 mg, 4.35 mmol) andtetra-n-butylammonium iodide (134 mg, 0.36 mmol) in DMF (10 mL) wasadded methoxyethoxymethyl chloride (248 μl, 2.18 mmol) at roomtemperature and the mixture was stirred for 4 h at the same temperatureand subsequently quenched by addition of methanol. The mixture was thendistributed between ether and brine and the organic layer dried oversodium sulfate, filtered and concentrated. The crude product waspurified by flash silica gel chromatography using a gradient of ethylacetate in hexanes to afford5-bromo-3-iodo-1-(2-methoxy-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(254 mg, 0.69 mmol, 74% yield) as a colorless solid (1:1 mixture withregioisomer5-bromo-3-iodo-2-(2-methoxy-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine).¹H-NMR (500 MHz, d₆-DMSO) isomer A (50%) S8.75 (d, 1H), 8.29 (d, 1H),5.78 (s, 1H), 3.61-3.63 (m, 2H), 3.37-3.39 (m, 2H), 3.17 (s, 3H); isomerB (50%) δ 8.74 (d, 1H), 8.28 (d, 1H), 5.77 (s, 1H), 3.61-3.63 (m, 2H),3.37-3.39 (m, 2H), 3.16 (s, 3H).

Step 2: Synthesis of5-bromo-1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine

A mixture of5-bromo-3-iodo-1-(2-methoxy-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(180 mg, 0.44 mmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (18 mg, 25 μmol) and 2-methoxyphenylboronic acid(82 mg, 0.51 mmol) in acetonitrile (3 mL) and 2 M aqueous solution ofsodium carbonate (3 ml) in a sealed vial was stirred at 60° C. for 2 h.The crude mixture was distributed between ethyl acetate and brine. Theaqueous phase was extracted with ethyl acetate (3×) and the combinedorganic phases were dried over sodium sulfate, filtered andconcentrated. The crude was then purified by flash silica gelchromatography using a gradient of ethyl acetate in hexanes to afford5-bromo-1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine(80 mg, 0.2 mmol, 46% yield) as a yellow oil. ¹H-NMR (500 MHz, d₆-DMSO)δ 8.70 (d, 1H), 8.40 (d, 1H), 7.62 (dd, 1H), 7.49 (ddd, 1H), 7.22 (d,1H), 7.09 (dt, 1H), 5.85 (s, 2H), 3.85 (s, 3H), 3.68-3.70 (m, 2H),3.39-3.41 (m, 2H), 3.18 (s, 3H). MS: m/z 316, 318 [MH⁺].

Step 3: Synthesis of3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid

A mixture of5-bromo-3-iodo-1/2-(2-methoxy-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(560 mg, 1.36 mmol, mixture of regioisomers), 2-methoxyphenylboronicacid (217 mg, 1.4 mmol) and1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (50 mg, 68 μmol) in acetonitrile (4 mL) and 2 Maqueous solution of sodium carbonate (2 mL) was stirred in a sealed vialat 70° C. for 105 min. The crude product was then distributed betweenethyl acetate and water. The aqueous phase was extracted ethyl acetate(3×) and the combined organic phases were washed with brine, dried oversodium sulfate, filtered and concentrated to afford5-bromo-1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine(780 mg, 75% pure, 1.12 mmol, 82% yield) as a crude dark oil. ¹H-NMR(500 MHz, d₆-DMSO) δ 8.70 (d, 1H), 8.40 (d, 1H), 7.62 (dd, 1H), 7.49(ddd, 1H), 7.22 (d, 1H), 7.09 (dt, 1H), 5.85 (s, 2H), 3.85 (s, 3H),3.68-3.70 (m, 2H), 3.39-3.41 (m, 2H), 3.18 (s, 3H). MS: m/z 316, 318[MH⁺].

A mixture of the above crude oil (1.12 mmol), 3-carboxyphenylboronicacid (259 mg (1.56 mmol) and1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (54 mg, 75 μmol) in acetonitrile (5 mL) and 2 Maqueous solution of sodium carbonate (5 mL) was irradiated in a PersonalChemistry Optimizer at 165° C. for 20 min. The crude product was dilutedwith acetonitrile and the organic phase was separated and concentrated.The residue was dissolved in potassium hydroxide in water (10% w/v, 15mL), washed with ethyl acetate (3×) and filtered through celite. Thefiltrate was then acidified to pH 3-4 by addition of concentratedaqueous hydrochloric acid and the precipitate was collected.Dichloromethane was then added to the precipitate, the insolublematerial filtered off and the filtrate concentrated to afford3-[1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoicacid (369 mg, 0.85 mmol, 57% yield) as a dark solid. ¹H-NMR (500 MHz,d₆-DMSO) δ 8.95 (d, 1H), 8.42 (d, 1H), 8.26 (t, 1H), 8.02 (dt, 1H), 7.98(dt, 1H), 7.68 (dd, 1H), 7.64 (t, 1H), 7.51 (ddd, 1H), 7.25 (d, 1H),7.12 (dt, 1H), 5.91 (s, 2H), 3.87 (s, 3H), 3.73-3.75 (m, 2H), 3.43-3.45(m, 2H), 3.21 (s, 3H). MS: m/z 432 [M−H⁻].

The resulting solid was dissolved in dichloromethane and PS-thiophenol(Argonaut Technologies) (1.4 mmol·g⁻¹, 1.2 g, 1.7 mmol,) andtrifluoroacetic acid (6 ml) were added. The resulting mixture was gentlystirred at 50° C. for 8.5 h. The resin was filtered off and washed withdichloromethane and ether. The combined filtrates were concentrated anddistributed between a saturated solution of sodium bicarbonate anddichloromethane. The aqueous phase was washed three times withdichloromethane and then acidified to pH 3-4 by addition of concentratedhydrochloric acid. The resulting aqueous phase was extracted three timeswith ethyl acetate. The combined ethyl acetate phases were washed withbrine, dried over sodium sulfate, filtered and concentrated to afford3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid(117 mg, 0.34 mmol, 40% yield, 25% over three steps) as a yellow solid.¹H-NMR (500 MHz, d₆-DMSO) δ 8.87 (d, 1H), 8.37 (d, 1H), 8.24 (t, 1H),8.02 (dt, 1H), 7.98 (dt, 1H), 7.68 (dd, 1H), 7.64 (t, 1H), 7.47 (ddd,1H), 7.23 (d, 1H), 7.11 (dt, 1H), 3.86 (s, 3H). MS: m/z 346 [MH⁺].

Step 4: Synthesis of{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-(4-methyl-piperazin-1-yl)-methanone

To a solution of3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid (25mg, 72 μmol) in anhydrous DMF (1.5 mL) was added PS-DCC resin (ArgonautTechnologies) (180 mg, 0.22 mmol, 1.21 mmol·g⁻¹) and N-methylpiperazine(9.6 μl, 86 μmol). The resulting mixture was stirred at 60° C. for 16 h.The resin was filtered off, washing with dichloromethane and ether andthe filtrate concentrated. The residue was dissolved in dichloromethaneand treated with a PS-trisamine resin (Argonaut Technologies) (20 mg).The resin was again removed by filtration and washed withdichloromethane and ether. The filtrate was concentrated and theresulting crude product was purified by reverse phase mass-triggeredHPLC using a gradient of acetonitrile in water containing 0.1% of formicacid to afford{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-(4-methyl-piperazin-1-yl)-methanone(1.7 mg, 4.0 μmol, 6% yield). ¹H-NMR (500 MHz, d₆-DMSO) δ 8.87 (d, 1H),8.37 (d, 1H), 8.24 (t, 1H), 8.02 (dt, 1H), 7.98 (dt, 1H), 7.68 (dd, 1H),7.64 (t, 1H), 7.47 (ddd, 1H), 7.23 (d, 1H), 7.11 (dt, 1H), 3.86 (s, 3H).MS: m/z 346 [MH⁺].

Other Compounds Prepared by Method 9:

TABLE 7 Structure

Method 10:

Step 1: Synthesis of3-[1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoicacid methyl ester

A mixture of5-bromo-1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine(535 mg, 1.36 mmol), 3-methoxycarbonylphenylboronic acid (258 mg, 1.43mmol) and 1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct (50 mg, 68 μmol) in acetonitrile (7 mL) and 2 Maqueous solution of sodium carbonate (7 mL) was irradiated in a PersonalChemistry Optimizer at 90° C. for 10 minutes. The resulting mixture wasdistributed between ethyl acetate and water. The aqueous phase wasextracted twice with ethyl acetate and the combined organic phases werewashed with brine, dried over sodium sulfate, filtered and concentrated.The crude was purified by flash silica gel chromatography using agradient of ethyl acetate and hexanes to afford3-[1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoicacid methyl ester (612 mg, 1.36 mmol, 100% yield) as a yellow oil.¹H-NMR (500 MHz, d₆-DMSO) δ 8.96 (d, 1H), 8.43 (d, 1H), 8.28 (t, 1H),8.07 (td, 1H), 7.99 (td, 1H), 7.67-7.69 (m, 2H), 7.51 (ddd, 1H), 7.25(d, 1H), 7.12 (dt, 1H), 5.92 (s, 2H), 3.90 (s, 3H), 3.87 (s, 3H),3.73-3.75 (m, 2H), 3.43-3.45 (m, 2H), 3.21 (s, 3H). MS: m/z 372 [MH⁺].

Step 2: Synthesis of3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acidmethyl ester

A solution of3-[1-(2-methoxy-ethoxymethyl)-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoicacid methyl ester (573 mg, 1.28 mmol) in dichloromethane (25 mL) wascooled down to 0-5° C. and boron trifluoride etherate (0.8 ml, 6.4 mmol)was added. The mixture was slowly warmed to room temperature and stirredfor 16 h. A yellow precipitate was formed was filtered off to afford3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acidmethyl ester (110 mg (0.29 mmol; 23% yield). ¹H-NMR (500 MHz, d₆-DMSO) δ8.88 (d, 1H), 8.39 (d, 1H), 8.27 (t, 1H), 8.06 (td, 1H), 7.99 (td, 1H),7.65-7.68 (m, 2H), 7.48 (ddd, 1H), 7.23 (d, 1H), 7.11 (dt, 1H), 3.90 (s,3H), 3.87 (s, 3H). MS: m/z 360 [MH⁺].

Step 3: Synthesis of{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-pyrrolidin-1-yl-methanone

3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acidmethyl ester (30 mg, 83 μmol) was dissolved in pyrrolidine (0.35 ml,4.15 mmol) and the mixture was stirred at 90° C. for 16 h. The mixturewas then concentrated and the crude product purified by flash silica gelchromatography using a gradient of 10% v/v of methanol in ethyl acetateto afford{3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-pyrrolidin-1-yl-methanoneas a yellow solid (22 mg, 55 μmol, 67% yield). ¹H-NMR (500 MHz, d₆-MeOH)δ 8.82 (d, 1H), 8.39 (d, 1H), 7.83 (t, 1H), 7.79 (td, 1H), 7.66 (dd,1H), 7.55-7.59 (m, 2H), 7.48 (ddd, 1H), 7.20 (d, 1H), 7.11 (dt, 1H),3.88 (s, 3H), 3.63 (t, 2H), 3.52 (t, 2H), 2.02 (m, 2H), 1.92 (m, 2H).MS: m/z 399 [MH⁺].

Method 11:

Step 1: Synthesis of{2-hydroxy-5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-pyridin-3-yl}-morpholin-4-yl-methanone

122 mg (0.25 mmol) of3-(2-methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine,150 mg (0.52 mmol) of(5-bromo-2-fluoro-pyridin-3-yl)-morpholin-4-yl-methanone, and 15 mg (18μmol) of 1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichlormethane adduct were placed in a Smith vial. 2 mL of acetonitrile,1 ml of water and 1 ml of a saturated aqueous solution of sodiumbicarbonate were added and the resulting mixture irradiated in aPersonal Chemistry Optimizer at 100° C. for 30 min. The resultingresidue was distributed between dichloromethane and a saturated aqueoussolution of sodium bicarbonate. The phases were separated and theaqueous phase was extracted twice with dichloromethane. The organicphases were combined, dried over sodium sulfate, filtered andconcentrated. The crude product was then purified by flash silica gelchromatography using a gradient of ethyl acetate containing 15% v/v ofmethanol and hexanes to afford 137 mg of a beige oil.

The oil was dissolved in 24 mL of a 1:1 mixture of dimethoxyethane andconcentrated aqueous hydrochloric acid. The mixture was heated to 55° C.for 1 h and then neutralized by addition of sodium bicarbonate. Theresulting mixture was distributed between ethyl acetate and water andthe aqueous phase was extracted three times with ethyl acetate. Thecombined organic phases were washed with brine, dried over sodiumsulfate and evaporated. The crude material was purified by reverse phasemass-triggered HPLC using a gradient of acetonitrile in water containing0.1% of formic acid to afford 12.3 mg (28 μmol, 11% yield) of{2-hydroxy-5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-pyridin-3-yl}-morpholin-4-yl-methanoneas an off-white solid upon lyophilization. ¹H-NMR (500 MHz, d₆-DMSO) δ13.79 (s, 1H), 12.25 (s, 1H), 8.77 (d, 1H), 8.26 (d, 1H), 8.00 (d, 1H),7.94 (d, 1H), 7.62 (dd, 1H), 7.46 (ddd, 1H), 7.22 (d, 1H), 7.09 (ddd,1H), 3.83 (s, 3H), 3.7-3.2 (m, 8H). MS: m/z 432 [MH⁺].

Method 12:

Step 1: Synthesis of (2-Amino-5-bromo-phenyl)-morpholin-4-yl-methanone

Into an 8 mL screw cap vial were added 5-bromoisatoic anhydride (0.200g, 0.826 mmol), anhydrous THF (5 mL), and morpholine (101 mg, 1.16mmol). The vial was sealed and placed in a heat block at 60° C. for 1.5h after which it was concentrated under vacuum. The crude product wastriturated with Et₂O/hexanes to afford 111 mg (94%) of(2-Amino-5-bromo-phenyl)-morpholin-4-yl-methanone as a tan solid. m/z285/287 [MH⁺].

Step 2: Synthesis of{2-Amino-5-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-morpholin-4-yl-methanone

Into a 5 mL Personal Chemistry microwave reaction vial were added3-(2-methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrrolo[2,3-b]pyridine(0.0498 g, 0.103 mmol),(2-Amino-5-bromo-phenyl)-morpholin-4-yl-methanone (0.0378 g, 0.132mmol), 1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichloromethane adduct (13.4 mg, 0.017 mmol), acetonitrile (1 mL) andsaturated aqueous NaHCO₃ (1 mL). The vial was sealed, purged with N₂,and irradiated in a Personal Chemistry Optimizer at 90° C. for 5 min.The layers were separated, and the aqueous phase was extracted 3× withEtOAc. The combined organic phase was treated with brine, dried(Na₂SO₄), filtered and concentrated. The crude product was dissolved in5 mL of a solution consisting of 1 part HClO₄ (70%, ACS) and 20 partsglacial acetic acid, and the solution was stirred at rt for 8 h. Thereaction mixture was concentrated under vacuum, and neutralized to pH 7with saturated NaHCO₃ followed by solid NaHCO₃. The quenched reactionmixture was partitioned between EtOAc and water, the layers wereseparated, and the aqueous phase was extracted 2× with EtOAc. Thecombined organic phase was treated with brine, dried (Na₂SO₄), filteredand concentrated. Purification by mass-triggered LC (positive mode, ESI)through a C-18 reverse-phase column (Thomson Instrument Co. ODS-A 100A,5μ, 50×21.3 mm, eluting at 20 mL/min with acetonitrile (containing 0.1%formic acid) and water (containing 0.1% formic acid) in a 5-95% gradientafforded the title compound, which upon lysophilization appeared as abrown viscous oil (12.9 mg, 29%). ¹H-NMR (500 MHz, d₆-DMSO) δ 13.71 (br.s, 1H), 8.74 (d, J=2.0 Hz, 1H), 8.16 (d, J=2.0 Hz, 1H), 7.62 (d, J=7.5Hz, 1H), 7.49 (dd, J=2.5, 8.0 Hz, 1H), 7.45 (m, 1H), 7.37 (d, J=2.0 Hz,1H), 7.20 (d, J=8.0 Hz, 1H), 7.08 (t, J=8.5 Hz, 1H), 6.81 (d, J=8.0 Hz,1H), 5.37 (s, 2H), 3.83 (s, 3H), 3.60 (m, 4H), 3.49 (m, 4H); MS: m/z430.1 [MH⁺].

Other Examples Prepared by Method 12:

TABLE 8 Structure

Method 13:

Step 1: Synthesis of2-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-thiazole-5-carboxylicacid

Into a 20 mL Personal Chemistry microwave reaction vial were added3-(2-Methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrrolo[2,3-b]pyridine(0.4992 g, 1.038 mmol), 2-Bromo-thiazole-5-carboxylic acid methyl ester(0.2592 g, 1.167 mmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichloromethane adduct (95.4 mg, 0.117 mmol), acetonitrile (5 mL) and 2Maqueous Na₂CO₃ (5 mL). The vial was sealed, purged with N₂, andirradiated in a Personal Chemistry Optimizer at 130° C. for 30 min. Thereaction mixture was diluted with EtOAc and acidified with acetic acidto pH 5. The layers were separated, and the aqueous phase was extracted5× with EtOAc. The combined organic phase was treated with brine, dried(Na₂SO₄), filtered and concentrated. The crude product was dissolved in10 mL of a solution consisting of 1 part HClO₄ (70%, ACS) and 20 partsglacial acetic acid, and the solution was stirred at rt for 4 h. Thereaction mixture was concentrated under vacuum, and neutralized to pH 7with saturated NaHCO₃ followed by solid NaHCO₃. The quenched reactionmixture was partitioned between EtOAc and water, the layers wereseparated, and the aqueous phase was extracted 2× with EtOAc. Theaqueous phase was then acidified to pH 4 with acetic acid and extracted5× with EtOAc. The combined organic extracts was treated with brine,dried (Na₂SO₄), filtered and concentrated. Trituration with Et₂Oafforded the title compound as a greenish-brown powder (0.296 g, 81%yield). ¹H-NMR (500 MHz, d₆-DMSO) δ 13.73 (br. s, 1H), 9.16 (s, 1H),8.71 (s, 1H), 8.40 (s, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.48 (t, J=8.0 Hz,2H), 7.24 (d, J=8.0 Hz, 1H), 7.10 (t, J=7.5 Hz 1H), 3.87 (s, 3H); MS:m/z 353.1 [MH⁺].

Other examples prepared by Method 13:

TABLE 9 Structure

Method 14:

Step 1: Synthesis of 3-Bromo-N,N-dimethyl-benzenesulfonamide

Into a 20 mL scintillation vial were added 3-Bromobenzenesulfonylchloride (0.301 g, 1.179 mmol) and anhydrous pyridine (5 mL). A 2Msolution of dimethylamine in THF (1.0 mL, 2.0 mmol) was added dropwise,and the reaction mixture was stirred at rt under N₂ for 5 h after whichit was concentrated under vacuum. The crude residue was partitionedbetween EtOAc and IM citric acid. The layers were separated, and theorganic phase was washed 3× with 1M citric acid, then treated withbrine, dried (Na₂SO₄), filtered and concentrated. Trituration with Et₂Oprovided 3-Bromo-N,N-dimethyl-benzenesulfonamide as a white powder(0.297 g, 96%). MS: m/z 263.9/265.9 [MH⁺].

Step 2: Synthesis of3-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-N,N-dimethyl-benzenesulfonamide

Into a 5 mL Personal Chemistry microwave reaction vial were added3-(2-Methoxy-phenyl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrrolo[2,3-b]pyridine(0.0496 g, 0.103 mmol), 3-Bromo-N,N-dimethyl-benzenesulfonamide (0.0417g, 0.143 mmol),1,1′-bis(diphenylphosphino)ferrocenepalladium(II)-dichloridedichloromethane adduct (13.9 mg, 0.017 mmol), acetonitrile (1 mL) andsaturated aqueous NaHCO₃ (1 mL). The vial was sealed, purged with N₂,and irradiated in a Personal Chemistry Optimizer at 90° C. for 15 min.The layers were separated, and the aqueous phase was extracted 3× withEtOAc. The combined organic phase was treated with brine, dried(Na₂SO₄), filtered and concentrated. The crude product was dissolved in5 mL of a solution consisting of 1 part HClO₄ (70%, ACS) and 20 partsglacial acetic acid, and the solution was stirred at rt for 1 h. Thereaction mixture was concentrated under vacuum, and neutralized to pH 7with saturated NaHCO₃ followed by solid NaHCO₃. The quenched reactionmixture was partitioned between EtOAc and water, the layers wereseparated, and the aqueous phase was extracted 2× with EtOAc. Thecombined organic phase was treated with brine, dried (Na₂SO₄), filteredand concentrated. Purification by mass-triggered LC (positive mode, ESI)through a C-18 reverse-phase column (Thomson Instrument Co. ODS-A 10A,5μ, 50×21.3 mm, eluting at 20 mL/min with acetonitrile (containing 0.1%formic acid) and water (containing 0.1% formic acid) in a 5-95% gradientafforded the title compound, which upon lysophilization appeared as alight yellow powder (10.4 mg, 25%). ¹H-NMR (500 MHz, d₆-DMSO) δ=13.91(br. s, 1H), 8.92 (d, J=2.0 Hz, 1H), 8.44 (d, J=2.0 Hz, 1H), 8.12 (m,1H), 8.01 (br.s, 1H), 7.76 (m, 2H), 7.67 (dd, J=2.0, 7.5 Hz, 1H), 7.45(m, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.09 (t, J=8.0 Hz, 1H), 3.85 (s, 3H),2.66 (s, 6H); MS: m/z 409.1 [MH⁺].

Other compounds prepared by Method 14:

TABLE 10 Structure

Method 15:

Step 1: Synthesis of3-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid

To a solution of5-bromo-3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(997 mg, 2.30 mmol) in acetonitrile (15 mL) and saturated aqueous NaHCO₃(10 mL) in a microwave vial was added 3-carboxyphenylboronic acid,pinacol ester (625 mg, 2.52 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II), complexwith dichloromethane (1:1) (94 mg, 0.12 mmol). The vial was capped,flushed with N₂, evacuated under vacuum, and heated in a microwave at90° C. for 1500 seconds. The acetonitrile was removed via rotaryevaporation. Ethyl acetate was added and then separated from the aqueouslayer. The organic layer was dark brown and LC/MS showed that theproduct remained in this layer. Concentrated down to a dark brown oiland redissolved in a 5% perchloric acid in acetic acid solution (10 mL).The reaction solution was stirred 4.5 hours at ambient temperature. Theperchloric acid was removed via rotary evaporation and then ethylacetate and H₂O were added. Sodium bicarbonate powder was added untilpH=3. The organic layer was separated, dried over Na₂SO₄, andconcentrated under vacuum to afford a brown powder (580 mg, 62% yield).¹H NMR (500 MHz, d₆-DMSO) δ 13.81 (br s, 1H), 8.81 (d, J=2.5 Hz, 1H),8.31 (d, J=2.5 Hz, 1H), 8.18 (s, 1H), 7.95 (d, J=7.5 Hz, 1H), 7.90 (d,J=7.5 Hz, 1H), 7.59 (m, 2H), 7.41 (t, J=7.0 Hz, 1H), 7.17 (d, J=8.0 Hz,1H), 7.04 (t, J=7.5 Hz, 1H), 3.80 (s, 3H). MS: m/e 346.1 (M+H⁺).

Step 2: Synthesis of{3-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-(4-pyrimidin-2-yl-piperazin-1-yl)-methanone

To a solution of3-[3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzoic acid (18mg, 0.05 mmol) in DMF (1 mL) was added HATU (20 mg, 0.05 mmol) and1-(2-pyrimidyl)piperazine (11 uL, 0.08 mmol). The reaction solution wasstirred for 16 hours at ambient temperature. The crude product wasextracted into ethyl acetate and washed with H₂O. The organic layer wasdried over Na₂SO₄, filtered, and adsorbed onto silica gel. Purificationby flash chromatography with a gradient of ethyl acetate (containing 10%MeOH) and hexanes afforded the title compound as a white powder (7.2 mg,28% yield). ¹H NMR (500 MHz, CD₃OD) δ 8.85 (d, J=2 Hz, 1H), 8.42 (d, J=2Hz, 1H), 8.35 (d, J=4.5 Hz, 2H), 7.83 (d, J=8 Hz, 1H), 7.79 (s, 1H),7.64 (m, 2H), 7.50 (m, 2H), 7.20 (d, J=8.5 Hz, 1H), 7.11 (t, J=8 Hz,1H), 6.64 (t, J=4.5 Hz, 1H), 3.96 (br s, 2H), 3.89 (s, 3H), 3.86 (br s,4H), 3.60 (br s, 2H). MS: m/z 492.1 (M+H⁺).

Other compounds made by Method 15:

TABLE 11 Structure

Method 16:

Step 1: Synthesis of5-bromo-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine

To a solution of5-bromo-3-(2-methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridine(260 mg, 0.60 mmol) in THF (3 mL) was added tetrabutylammonium fluoride(6 mL of 1 M in THF solution, 6.00 mmol) and molecular sieves. Thesolution was heated under reflux (70° C.) for 4 hours without stirring.The solution was cooled to ambient temperature and acidified to pH=5 byadding dropwise a dilute solution of acetic acid in MeOH. The solutionwas filtered and the filtrate was concentrated via rotary evaporation.The material was extracted into EtOAc and washed 3×H₂O. The organiclayer was dried over Na₂SO₄ and concentrated down to afford5-bromo-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine as an orangesolid (177 mg, 97% yield). MS: m/z 303.9, 305.9 [M+H⁺]. The material wasused directly in step 2 without further purification.

Step 2: Synthesis of4-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-N-methyl-benzamide

To a solution of 5-bromo-3-(2-methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridine(26 mg, 0.085 mmol) in acetonitrile (1 mL) and saturated aqueous NaHCO₃(1 mL) in a microwave vial was added4-(N-methylaminocarbonyl)phenylboronic acid (17 mg, 0.094 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II), complexwith dichloromethane (1:1) (3.5 mg, 0.004 mmol). The vial was capped,flushed with N₂, evacuated under vacuum, and heated in a microwave at130° C. for 1800 seconds. The material was extracted into EtOAc and theorganic layer was dried over Na₂SO₄. The material was adsorbed onto SiO₂and purified by flash chromatography in a EtOAc (containing 10% MeOH)and hexane gradient. The clean fractions were concentrated via rotaryevaporation to afford the title compound as a white powder (5.9 mg, 20%yield). ¹H NMR (500 MHz, CD₃OD) δ 8.85 (s, 1H), 8.41 (s, 1H), 7.94 (d,J=8.5 Hz, 2H), 7.78 (d, J=8.5 Hz, 2H), 7.67 (d, J=7.5 Hz, 1H), 7.48 (t,7.0 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 7.11 (t, J=7.0 Hz, 1H), 3.89 (s,3H), 2.95 (s, 3H). MS: m/z 359.1 [M+H⁺].

Other compounds prepared by Method 16:

TABLE 12 Structure

Method 17:

Step 1: Synthesis of3-(2-Methoxy-phenyl)-5-(3-pyrrolidin-1-ylmethyl-phenyl)-1H-pyrazolo[3,4-b]pyridine

To a solution of3-[3-(2-Methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzaldehyde(23 mg, 0.050 mmol) and Pyrrolidine (5 ul, 0.082 mmol) in 1.5 mldichloethane was added 3 ul of AcOH. The mixture was stirred at roomtemperature for 30 mins, and then to the mixture was added sodiumtrioxyacetylborohydride (22 mg, 0.10 mmol) in one portion. The reactionwas continued at room temperature for another 2 hrs the mixture was thenconcentrated to yield the SEM product which was treated with a solutionof 5% perchlorate in acetic acid (2 mL) at room temperature for 1 hr.Solvents were evaporated, the residue was neutralized with sodiumbicarbonate powder and then purified by flash silica gel chromatographyusing ethyl acetate then a mixture of EtOAc/DCM/MeOH/NH4OH (4/4/1/0.05)to afford3-(2-Methoxy-phenyl)-5-(3-pyrrolidin-1-ylmethyl-phenyl)-1H-pyrazolo[3,4-b]pyridine(8.50 mg, 44% yield) as a white solid. ¹H NMR (500 MHz, CD₃OD) δ 1.89(d, J=3 Hz, 4H), 2.76 (d, J=1.5 Hz, 4H), 3.89 (s, 2H), 3.9 (s, 2H), 7.11(t, J=1 Hz, 1H), 7.22 (d, J=8 Hz, 1H), 7.42 (t, J=7 Hz, 1H), 7.47 (d,J=1.5 Hz, 1H), 7.48 (d, J=2 Hz, 1H), 7.51 (d, J=2.5 Hz, 1H), 7.65 (t,J=6.75 Hz, 1H), 7.72 (s, 1H), 8.39 (d, J=2.5 Hz, 1H), 8.83 (d, J=2 Hz,1H). MS: m/z 385 [MH⁺].

Other Compounds Prepared by Method 17:

TABLE 13 Structure

Method 18:

Step 1: Synthesis of 5-Bromo-3-trimethylsilanylethynyl-pyrazin-2-ylamine

To a solution of 3,5-Dibromo-pyrazin-2-ylamine (3.00 g, 11.86 mmol) inDMF (35 ml) was added triethylamine (16 ml), then tetrakistriphenylphinepalladium (0) (685 mg, 0.59 mmol) and copper(I) iodide (271 mg, 1.42mmol) were added sequentially. Finally trimethylsilylacetylene (2.0 ml,14.3 mmol) was added dropwise. The reaction mixture was stirred at 120°C. for 30 minutes and then directly adsorbed onto silica gel.Purification by flash chromatography on silica gel with a gradient ofethyl acetate/hexane afforded the title compound (2.30 g, 71% yield) asyellow oil. MS: m/z 270.0/272.0 [MH⁺].

Step 2: Synthesis ofN-(5-Bromo-3-trimethylsilanylethynyl-pyrazin-2-yl)-acetamide

To a solution of 5-Bromo-3-trimethylsilanylethynyl-pyrazin-2-ylamine(2.30 g, 8 mmol) in anhydrous THF (35 ml) and pyridine (1.62 ml, 20.0mmol) was added acetyl chloride (682 μl, 9.6 mmol). The mixture wasstirred at room temperature overnight, and then stirred at 60° C. for 5hours. Solvents were removed in vacuum and the resulting brown residuewas purified by silica gel chromatogrpahy with a gradient of ethylacetate/hexane to afford the title compound (474 mg, 1.52 mmol) as lightyellow as off white solid. MS: m/z 311.9/313.9 [MH⁺].

Step 3: Synthesis of 2-Bromo-5H-pyrrolo[2,3-b]pyrazine

To a solution ofN-(5-Bromo-3-trimethylsilanylethynyl-pyrazin-2-yl)-acetamide (474 mg,1.52 mmol) in THF (4 ml) was added dropwise a 1 M solution oftetra-n-butyl ammonium fluoride in THF (3.3 ml, 3.3 mmol). Afterstirring at reflux for 15 hours, the reaction mixture was concentratedin vacuum and water added. The aqueous layer was extracted three timeswith dichloromethane and the combined extracts were directly adsorbed onsilica gel. Purification by silica gel chromatography with a gradient ofethyl acetate/hexanes afforded the title compound (130 mg, 43% yield) asa yellow solid. ¹H-NMR (500 MHz, d₆-DMSO) δ 12.38 (s br, 1H), 8.38 (s,1H), 7.95 (d, 3.5 Hz, 1H), 6.61 (d, 3.5 Hz, 1H). MS: m/z 197.9/199.9[MH⁺].

Step 4: Synthesis of 2-Bromo-7-iodo-5H-pyrrolo[2,3-b]pyrazine

To a solution of 2-Bromo-5H-pyrrolo[2,3-b]pyrazine (258 mg, 1.3 mmol) inacetone (5 ml) was added N-iodosuccinimide (324 mg, 1.44 mmol) in oneportion. The reaction mixture was stirred at room temperature for 45minutes. The resulting precipitate was filtered off, washed with aminimal amount of acetone, and dried in vacuum to give the titlecompound as a light brown solid ¹H-NMR (500 MHz, d₆-DMSO) δ 12.81 (s br,1H), 8.40 (s, 1H), 8.19 (d, 3.0 Hz, 1H). MS: m/z 323.8/325.8 [MH⁺].

Step 5: Synthesis of2-Bromo-7-iodo-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine

To a suspension of 2-Bromo-7-iodo-5H-pyrrolo[2,3-b]pyrazine (290 mg,0.895 mmol) in THF (5 ml) was added NaH (60%, 43 mg, 1.08 mmol) in oneportion at 0° C. The resulting mixture was stirred for 20 minutes beforea solution of para-toluenesulfonyl chloride (188 mg, 0.98 mmol) in THF(2 mL) was added. The reaction mixture was then stirred at roomtemperature for 3 hours. Solvents were removed and the resulting darkbrown residue washed with aqueous KOH, water and dried to afford thetitle compound (423 mg, 99% yield) as a light brown solid. ¹H-NMR (500MHz, d₆-DMSO) δ 8.60 (d, 11.5 Hz, 1H), 7.99 (d, 11.5 Hz, 2H), 7.44 (d,7.5 Hz, 2H), 2.34 (s, 3H). MS: m/z 477.8/479.8 [MH⁺].

Step 6: Synthesis of2-Bromo-7-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine

A 50-ml round bottom flask was charged with2-Bromo-7-iodo-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine (423 mg,0.885 mmol), 2-methoxyphenylboronic acid (148 mg (0.973 mmol) anddichlorobis(triphenylphosphino)palladium(II) (31 mg, 0.04 mmol). To thismixture was added acetonitrile (10 mL) and a 2 M aqueous solution ofsodium bicarbonate (5 mL). The reaction mixture was stirred at 40° C.for 1 hour, then 55° C. for another hour. The crude reaction mixture wasdistributed between ethyl acetate and a saturated aqueous solution ofsodium bicarbonate. The aqueous phase was then extracted with ethylacetate and the combined organic phases were dried over sodium sulfate,filtered and concentrated. The crude product was then purified by flashsilica gel chromatography using a gradient of ethyl acetate in hexanesto afford2-Bromo-7-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine(139 mg, 34% yield) as a light yellow solid; the bis-addition product2,7-Bis-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine(213 mg, 50% yield) was also obtained. MS: m/z 457.9/460.0 [MH⁺]; MS:m/z 486.1 [MH⁺] (bis-addition product).

Step 7: Synthesis of3-[7-(2-Methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-benzoic acid

A mixture of2-Bromo-7-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine(70 mg, 0.15 mmol),3-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzoic acid (57 mg,0.23 mmol) and dichlorobis(triphenylphosphino)palladium(II) (5.4 mg,0.008 mmol) in acetonitrile (2 mL) and aqueous solution of sodiumcarbonate (2M, 2 mL) was irradiated in a Personal Chemistry Optimizer at95° C. for 20 minutes. The crude reaction mixture was distributedbetween dichloromethane and a saturated aqueous solution of sodiumbicarbonate. The aqueous phase was then extracted with dichloromethaneand the combined organic phases were dried over sodium sulfate, filteredand concentrated.

The crude brown residue was then dissolved in MeOH (2 mL) and 5N KOH(150 μL) was added. The mixture was then stirred at 40° C. for 2 hoursbefore the solvents were removed. The resulting yellow residue waswashed with diluted HCl (2 ml), water, and then dried to afford thecrude acid3-[7-(2-Methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-benzoic acid,which was used directly in Step 8. MS: m/z 346.0 [MH⁺].

Step 8: Synthesis of[4-(2-Dimethylamino-ethyl)-piperazin-1-yl]-{3-[7-(2-methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-phenyl}-methanone

To a mixture of3-[7-(2-Methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-benzoic acid (35mg, 0.1 mmol), EDCI (77 mg, 0.40 mmol), HBTU (3.8 mg, 0.01 mmol) anddiisopropylethylamine (175 μl, 1.0 mmol) in DMF (1 mL) was added2-(dimethylamino)ethylpiperazine (55 μl, 0.3 mmol). The mixture wasstirred at 70° C. for 5 hours before the solvents were removed. Theresulting yellow oil was washed with water, dissolved in DMSO andpurified on reverse phase HPLC to afford the title compound (8.6 mg, 12%over 2 steps) as yellow solid. MS: m/z 485.2 [MH⁺].

Other compounds prepared by method 18:

TABLE 14 Structure

Method 19:

Step 1: Synthesis of3-[7-(2-Methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-N,N-dimethyl-benzamide

A mixture of2-Bromo-7-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine(30 mg, 0.065 mmol), 3-dimethylaminocarbonylphenyl boronic acid (25.3mg, 0.130 mmol) and dichlorobis(triphenylphosphino)palladium(II) (2.5mg, 0.003 mmol) in acetonitrile (1 mL) and aqueous solution of sodiumbicarbonate (2M, 1 mL) was irradiated in a Personal Chemistry Optimizerat 90° C. for 15 minutes. The crude reaction mixture was distributedbetween dichloromethane and a saturated aqueous solution of sodiumbicarbonate. The aqueous phase was then extracted with dichloromethaneand the combined organic phases were dried over sodium sulfate, filteredand concentrated. The crude brown residue was then dissolved in MeOH (5mL), 5N KOH (50 μL) and the mixture was stirred at room temperature for75 minutes. Removal of the solvents resulted in a yellow residue, whichwas then purified by flash silica gel chromatography using a gradient ofethyl acetate in hexanes, then 10% MeOH in EtOAc to afford3-[7-(2-Methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]-N,N-dimethyl-benzamide(13.0 mg, 54% yield) as a pale yellow solid. ¹H-NMR (500 MHz, CD3OD) δ8.89 (m, 1H), 8.80 (m, 1H), 8.40 (m, 1H), 8.25 (m, 2H), 7.62 (m, 1H),7.50 (m, 1H), 7.25 (m, 1H), 7.10 (m, 2H), 3.97 (m, 3H), 3.17 (m, 3H),3.10 (m, 3H). MS: m/z 373.1 [MH⁺].

Other Compounds Prepared by Method 19:

TABLE 15 Structure

Method 20:

Step 1: Synthesis of2,7-Bis-(2-methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazine

To a solution of2,7-Bis-(2-methoxy-phenyl)-5-(toluene-4-sulfonyl)-5H-pyrrolo[2,3-b]pyrazine(200 mg, 0.412 mmol) in MeOH (5 mL) was added a solution of NaOH (66 mg,1.65 mmol) in water (200 μL). The mixture was stirred at roomtemperature for 3.5 hours before the solvents were removed. Theresulting yellow residue was then purified by flash silica gelchromatography using a gradient of ethyl acetate in hexanes to afford2,7-Bis-(2-methoxy-phenyl)-5H-pyrrolo[2,3-b]pyrazine (43 mg, 32% yield)as a pale yellow solid. ¹H-NMR (500 MHz, CD3OD) δ 12.21 (s br, 1H), 8.80(dd, 6.0 Hz, 1.75 Hz, 1H), 8.69 (s, 1H), 7.76 (dd, 6.0 Hz, 1.75 Hz, 1H),7.43 (dt, 7.5 Hz, 1.75 Hz, 1H), 7.20 (m, 2H), 7.13 (m, 2H), 7.04 (t, 7.3Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H). MS: m/z 332.1 [MH⁺].

Method 21:

Step 1: Synthesis of 3-amino-6-iodo-pyrazine-2-carboxylic acid methylester

Methyl 3-amino-2-pyrazinecarboxylate (10 g, 65.3 mmol) andN-iodosuccinimide (24 g, 106.7 mmol) were dissolved in anhydrous DMF(150 mL) and the mixture was stirred at 70° C. for 15 hours under anitrogen atmosphere. The mixture was then cooled to room temperature anda saturated aqueous solution of sodium thiosulfate (400 mL) was added.The suspension was sonicated for 15 minutes, concentrated under vacuumand dispersed in water. The crude product was filtered off and washedwith cold ethanol. The residue was crystallized from ethanol, usingdecolorizing charcoal to afford 3-amino-6-iodo-pyrazine-2-carboxylicacid methyl ester (11.2 g, 61% yield) as orange needles. 1H-NMR(d6-DMSO) δ: 8.57 [1H] s, 7.59 [2H] s, br, 3.93 [3H] s. MS: m/z 280[MH⁺].

Step 2: Synthesis of 3-amino-6-iodo-pyrazine-2-carboxylic acid

3-amino-6-iodo-pyrazine-2-carboxylic acid methyl ester (45 g, 161 mmol)was dissolved in 750 ml of THF. 90 ml of water and 40 ml of a 4 Msolution of lithium hydroxide in water was added. The mixture wasstirred at room temperature for 2 hours or until TLC analysis showedonly baseline material. A solution of 10% citric acid in water was addedto adjust the pH to about 3-4. The mixture was diluted withdichloromethane and the organic phase was separated. The aqueous layerwas extracted three times with dichloromethane and the combined organicphases were dried over sodium sulfate and evaporated. The residue wasdried in vacuo to afford 37.0 g (140 mmol; 87% yield) of3-amino-6-iodo-pyrazine-2-carboxylic acid as a yellow powder. ¹H-NMR(d6-DMSO) δ: 11.70 s, weak, 8.44 [1H] s, 7.50 [2H] s, br. MS: m/z 266[MH⁺].

Step 3: Synthesis of 3-amino-6-iodo-pyrazine-2-carboxylic acidmethoxy-methyl-amide

27.50 g (0.104 mol) of 3-amino-6-iodo-pyrazine-2-carboxylic acid, 63.0 g(0.125 mol) of PyBOP (1-benzotriazolyloxy-tris(pyrrolidino)phosphoniumhexafluorophosphate) and 18.70 g (0.193 mol) ofN,O-dimethylhydroxylamine hydrochloride were placed in a nitrogenflushed flask and then dissolved in a mixture of 100 ml of anhydrous DMFand 27 ml of N,N-di-iso-propylethylamine. The mixture was heated to 80°C. for 16 hours. The solvent was evaporated at 50-60° C. under reducedpressure to afford a dark oil. The oil was extracted three to four timeswith 300 ml of toluene. The toluene phases were combined and evaporated.The resulting oil was purified via chromatography on silica gel using agradient of ethyl acetate in hexanes to afford 18.40 g (59.72 mmol; 58%)of 3-amino-6-iodo-pyrazine-2-carboxylic acid methoxy-methyl-amide as ayellow solid. ¹H-NMR (d6-DMSO) δ: 8.28 [1H] s, 6.78 [2H] s, 3.66 [3H] s,3.25 [3H] s. MS: m/z 309 [MH⁺].

Step 4: Synthesis of (3-amino-6-iodo-pyrazin-2-yl)-phenyl-methanone

8.00 g (25.97 mmol) of 3-amino-6-iodo-pyrazine-2-carboxylic acidmethoxy-methyl-amide was dissolved in 100 ml of anhydrous THF undernitrogen. The solution was cooled to 55° C. and 27 ml of a 3 M solutionof phenylmagnesium bromide in ether was added. The mixture was allowedto warm to 10° C. and a solution of 10% citric acid in water was added.The mixture was diluted with dichloromethane and the phases wereseparated. The aqueous phase was extracted three times withdichloromethane and the combined organic phases were dried over sodiumsulfate and evaporated. The resulting solid was crystallized fromethanol to afford 5.74 g (17.66 mmol; 68%) of(3-amino-6-iodo-pyrazin-2-yl)-phenyl-methanone as yellow-orangecrystals. ¹H-NMR (d6-DMSO) δ: 8.52 [1H] s, 7.94 [2H] s, br, 7.85 [2H] d,7.62 [1H] t, 7.52 [2H] t. MS: m/z 326 [MH⁺].

Step 5: Synthesis of5-iodo-3-(2-methoxy-1-phenyl-vinyl)-pyrazin-2-ylamine

2.52 g (12.6 mmol) of potassium bis(trimethylsilyl)amide was dissolvedin 150 ml of anhydrous THF under nitrogen. 3.80 g (11.1 mmol) ofmethoxymethyltriphenylphosphonium chloride was added and the mixturestirred at room temperature for 1 hour. To the resulting mixture wasadded 2.50 g (7.69 mmol) of(3-amino-6-iodo-pyrazin-2-yl)-phenyl-methanone and the reaction mixturewas stirred at room temperature for 1 hour. The resulting reactionmixture was heated to reflux for 20 hours. After cooling the mixture wasdiluted with dichloromethane and washed with a saturated aqueoussolution of ammonium chloride in water and dried over sodium sulfate.The solvent was evaporated and the residue purified by chromatography onsilica gel using a gradient of ethyl acetate in hexanes to afford 1.942g (5.50 mmol; 72%) of partially resolved E- andZ-5-iodo-3-(2-methoxy-1-phenyl-vinyl)-pyrazin-2-ylamine as a pale yellowsolid. ¹H-NMR (d6-DMSO) δ: isomer A 8.12 [1H] s, 7.33-7.26 [4H] m, 7.20[1H] m, 6.66 [1H] s, 6.00 [2H] s, br, 3.81 [3H] s; isomer B 8.09 [1H] s,7.27 [2H] t, 7.17 [1H] t, 7.11 [2H] d, 6.98 [1H] s, 6.24 [2H] s, br,3.76 [3H] s. MS: m/z 354 [MH⁺].

Step 6: Synthesis of 5-iodo-3-phenyl-1H-pyrrolo[2,3-b]pyrazine

780 mg of 5-iodo-3-(2-methoxy-1-phenyl-vinyl)-pyrazin-2-ylamine (E-,Z-form or mixture) was dispersed in 40 ml of a 1:1 mixture of dilutehydrochloric acid in water (approx. 1-2 N) and ethanol. The mixture washeated to reflux for 2 hours. Ice was added to the resulting suspensionand the precipitate filtered off to afford 480 mg of5-iodo-3-phenyl-1H-pyrrolo[2,3-b]pyrazine as a pale yellow powder. Thefiltrate was made basic by addition of sodium bicarbonate and extractedthree times with dichloromethane. The combined extracts were dried oversodium sulfate and evaporated. The residue was crystallized from ethanolto afford 76 mg of 5-iodo-3-phenyl-1H-pyrrolo[2,3-b]pyrazine asgreen-brown crystalline needles for a combined yield of 556 mg (78%).¹H-NMR (d6-DMSO) δ: 12.56 [1H] s, br, 8.53 [1H] s, 8.46 [1H] d, 8.14[2H] d, 7.45 [2H] dd, 7.25 [1H] dd. MS: m/z 322 [MH⁺].

Step 7: Synthesis of5-(3,4-dimethoxy-phenyl)-3-phenyl-1H-pyrrolo[2,3-b]pyrazine

50 mg (0.16 mmol) of 5-iodo-3-phenyl-1H-pyrrolo[2,3-b]pyrazine, 38 mg(0.20 mmol) of 3,4-dimethoxyphenylboronic acid and 6 mg (5 mol %) ofdichlorobis(triphenylphosphino)palladium(II) were placed in a vial and 1ml of acetonitrile and 1 ml of a 2 M aqueous solution of sodiumcarbonate were added and the mixture irradiated in a Personal Chemistry®microwave reactor to 165° C. for 1200 sec. The resulting mixture wasdistributed between 15 ml of a saturated aqueous solution of sodiumbicarbonate and 75 ml of dichloromethane. The organic phase was driedover sodium sulfate and evaporated. The crude was purified via flashchromatography on silica gel using a gradient of methanol indichloromethane. The product isolated was crystallized from hot ethanolto afford 14 mg (43 μmol, 27% yield) of5-(3,4-dimethoxy-phenyl)-3-phenyl-1H-pyrrolo[2,3-b]pyrazine as an orangepowder. ¹H-NMR (d6-DMSO) δ: 12.30 [1H] s, 8.92 [1H] s, 8.43 [1H] s, 8.35[2H] d, 7.80 [1H] (m), 7.79 [1H] d (m), 7.46 [2H] dd, 7.25 [1H] dd (d),7.13 [1H] d, 3.92 [3H] s, 3.84 [3H] s. MS: m/z 332 [MH⁺].

Other Compounds Prepared by Method 21:

TABLE 16 Structure

Method 22:

Step 1: Synthesis of5-(morpholin-4-yl)-3-phenyl-1H-pyrrolo[2,3-b]pyrazine

25 mg (80 μmol) of 5-iodo-3-phenyl-1H-pyrrolo[2,3-b]pyrazine wasdissolved in 1 ml of morpholine. 200 μl of glacial acetic acid was addedand the mixture heated in a Personal Chemistry® microwave reactor to250° C. for 2400-4800 sec. The crude was purified by flashchromatography on silica gel without prior workup using a gradient ofethyl acetate in hexanes to afford 13 mg (46 μmol, 58% yield) of2-morpholin-4-yl-7-phenyl-1H-pyrrolo[2,3-b]pyrazine as a beige solid.¹H-NMR (d6-DMSO) δ: 11.89 [1H] s, 8.19 [1H] s, 8.18 [2H] d, 7.39 [2H]dd, 7.17 [1H] dd, 3.80 [4H] t, 3.52 [4H] t. MS, m/z: 281 [MH+].

TABLE 17 Structure

Method 23:

Step 1: Synthesis of 4-(5-iodo-1H-pyrrolo[2,3-b]pyrazin-3-yl)-phenol

80 mg of5-iodo-3-{2-methoxy-1-[4-(tetrahydro-pyran-2-yloxy)-phenyl]-vinyl}-pyrazin-2-ylaminewas dispersed in 70 ml of dilute (1-2 N) aqueous hydrochloric acid.Methanol was added to dissolve the starting material (10-20% v/v) toafford a clear solution. 0.5 ml of concentrated hydrochloric acid wasadded and the mixture was heated to reflux for 7 hours. The mixture wascooled to room temperature and allowed to stir for 16 hours. The mixturewas neutralized by addition of sodium bicarbonate and water added asneeded to keep salts in solution. The resulting mixture was extractedfour times with dichloromethane and the combined organic phases were,dried over sodium sulfate and evaporated to afford 428 mg (1.27 mmol,85% yield) of 4-(5-iodo-1H-pyrrolo[2,3-b]pyrazin-3-yl)-phenol as anorange solid of sufficient purity (>85%), which could be recrystallizedfrom dichloromethane-ethyl acetate to afford 195 mg (578 μmol, 39%yield) of pure 4-(5-iodo-1H-pyrrolo[2,3-b]pyrazin-3-yl)-phenol asyellow-orange crystals. ¹H-NMR (d6-DMSO) δ: 12.37 [1H] (d), 9.43 [1H] s,8.49 [1H] s, 8.26 [1H] d, 7.92 [2H] d, 8.85 [2H] d. MS: m/z 338 [MH⁺].

Step 2: Synthesis of 4-{5-[3-methoxy-4-hydroxyphenyl]-1H1-pyrrolo[2,3-b]pyrazin-3-yl}-phenol

84 mg (0.25 mmol) of 4-(5-iodo-1H-pyrrolo[2,3-b]pyrazin-3-yl)-phenol,120 mg (0.33 mmol) of2-[3-methoxy-4-(4-methoxy-benzyloxy)-phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolaneand 15 mg (8 mol %) of dichlorobis(triphenylphosphino)palladium(II) wereplaced in a vial and 1.5 ml of acetonitrile and 1.5 ml of a 2 M aqueoussolution of sodium carbonate were added. The mixture was irradiated in aPersonal Chemistry® microwave reactor to 165° C. for 1200 sec. Theresulting mixture was distributed between dichloromethane and asaturated aqueous solution of sodium bicarbonate. The aqueous layer wasextracted twice with dichloromethane and the combined organic phaseswere dried over sodium sulfate and evaporated. The crude was purified byflash chromatography on silica gel using a gradient of ethyl acetate inhexanes. The resulting intermediate was dissolved in 120 ml ofdichloromethane and 1.5 g (2.12 mmol) of PS-thiophenol (ArgonautTechnologies) was added. To this was added 2 ml of trifluoroacetic acidand the mixture stirred at room temperature for 1 hour. The resin wasfiltered off and washed with dichloromethane. The filtrate was washedwith a saturated aqueous solution of sodium bicarbonate. The phases wereseparated and the aqueous layer extracted twice with ethyl acetate. Allorganic phases were combined, dried over sodium sulfate and evaporated.The residue was heated up with acetonitrile, cooled down to roomtemperature and the supernatant was removed. The residue was dried invacuo to afford 15 mg (45 μmol, 18% yield) of4-{5-[3-methoxy-4hydroxyphenyl]-1H-pyrrolo[2,3-b]pyrazin-3-yl}-phenol asa beige powder. ¹H-NMR (d6-DMSO) δ: 12.06 [1H] d, 9.35 [1H] s, 9.30 [1H]s, 8.83 [1H] s, 8.20 [1H] d, 8.12 [2H] d (m), 7.75 [1H] d, 7.65 [1H] dd,6.93 [1H] d, 6.85 [2H] d (m), 3.91 [3H] s. MS, m/z: 334 [MH⁺].

Method 24

Step 1: Synthesis of methyl3-(3-(2-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzimidate

HCl gas was bubbled through a suspension of3-[3-(2-Methoxy-phenyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-benzonitrile(40 mg, 0.088 mmol) in 2.5 ml of anhydrous MeOH for 3 minutes at 0° C.After stirring for 23 hours at room temperature, ether (10 mL) was addedand precipitation occurred. The solid was collected after filtration anddried to afford methyl3-(3-(2-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzimidate as ayellow solid.

Step 2: Synthesis ofC-{3-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-C-morpholin-4-yl-methyleneamine

A solution of methyl3-(3-(2-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzimidate fromStep in MeOH (1.0 mL) was added morphiline (15.3 mg, 0.176 mmol) andtriethylamine (90 mg, 0.88 mmol), the mixture was stirred at roomtemperature for 3 days. The solvent was then removed and the crudeproduct purified by reverse phase HPLC to affordC-{3-[3-(2-Methoxy-phenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl]-phenyl}-C-morpholin-4-yl-methyleneamine(3.7 mg, 10% yield two steps) as a white solid. ¹H-NMR (500 MHz, CD3OD)δ 8.86 (d, 2 Hz, 1H), 8.45 (d, 2 Hz, 1H), 8.37 (br s, 1H), 8.02 (m, 1H),7.96 (t, 1.8 Hz, 1H), 7.76 (t, 7.8 Hz, 1H), 7.66 (dd, 1.8 Hz, 7.8 Hz,1H), 7.63 (m, 1H), 7.49 (m, 1H), 7.21 (d, 8 Hz, 1H), 7.12 (dt, 1 Hz, 7.8Hz, 1H), 3.95 (m, 2H), 3.88 (s, 3H), 3.82 (m, 2H) 3.78 (m, 2H), 3.59 (m,2H). MS: m/z 414.1 [MH⁺].

Other Compounds Prepared by Method 24:

TABLE 18 Structure

Bioassays:

Kinase assays known to those of skill in the art may be used to assaythe inhibitory activities of the compounds and compositions of thepresent invention. Kinase assays include, but are not limited to, thefollowing examples.

Although the first of these examples uses the kinase domain of a mutantform of Abl T3151 (“Abl T3151 KD”), the kinase assays may use variousforms of mutant and wild type enzymes, including, for example, theentire protein, the kinase domain, or a portion thereof (e.g. AblY393F). The kinases used in the assays may also be of varyingphosphorylation states. In the c-Abl example, a mutant kinase at a zerophosphorylation state was used.

c-Abl Pyruvate Kinase/Lactate Dehydrogenase Coupled Enzyme Assay

In the c-Abl Pyruvate Kinase (PK)/Lactate Dehydrogenase (LDH) CoupledAssay the protein kinase dependant phosphorylation of a substratepeptide was coupled to the oxidation of NADH. The oxidation of NADH toNAD+ was detected by monitoring a decrease in absorbance at 340 nm.

Materials: Abl substrate peptide=EAIYAAPFAKKK-OH (Biopeptide, San Diego,Calif.); βNADH (Sigma Cat#N-8129, FW=709.4); 2M MgCl₂; 1M HEPES buffer,pH 7.5; Phosphoenolpyruvate (PEP) (Sigma Cat#P-7002, FW=234); Lactatedehydrogenase (LDH) (Worthington Biochemical Cat#2756); Pyruvate Kinase(PK) (Sigma Cat#P-9136); ATP (Sigma Cat#A-3377, FW=551); Greiner384-well UV star plate; and purified and unphosphorylated T315I Ablkinase domain.

Stock Solutions: 10 mM NADH (7.09 mg/ml in miliQH₂O) made fresh daily;10 mM Abl substrate peptide (13.4 mg/ml in miliQH₂O) stored at −20° C.;100 mM HEPES buffer, pH 7.5 (5 ml 1M stock+45 ml miliQH₂O); 100 mM MgCl₂(5 ml 2M MgCl₂+95 ml dH₂O); 100 mM PEP (23.4 mg/ml in dH₂O) stored at−20° C.; 10 mM ATP (5.51 mg/ml in dH₂O) stored at −20° C. (diluted 50 μlinto total of 10 ml miliQH₂O daily=50 μM ATP working stock); 1000 U/mlPK (U/mg varies with lot) flash-frozen under liquid N₂ and stored at−80° C.; and 1000 U/ml LDH (U/mg varies with lot) flash-frozen underliquid N₂ and stored at −80° C.

Standard Assay Setup for 384-well format (50 μl reaction): 300 μM NADH;10 mM MgCl₂; 2 mM PEP; 45 U/ml PK; 60 U/ml LDH; 200 μM Abl substratepeptide; 2.5 μl test compound (in DMSO); 2 μg/ml Abl kinase domain; 10μM ATP; 100 mM HEPES buffer. Positive controls contained DMSO with notest compound. Negative controls contained 5 μl of 0.5M EDTA (50 mM inthe assay). The dephosphorylated form of the c-Abl T3151 mutant was usedin the biochemical screening assays. The kinase reaction was initiatedat time t=0 by the addition of ATP.

Activity was measured by following the time-dependent loss of NADH byabsorbance spectroscopy at 340 nm. The linear portion of the resultingprogress curve was then analyzed by linear regression to get theactivity in absorbance units/time, reported as the slope of that bestfit line (moles/unit time can be calculated from using molar extinctioncoefficient for NADH at 340 nm, 6250M⁻¹ cm⁻¹).

Data was evaluated using the equation: Z′=1−[3*(σ₊+σ⁻)/|μ₊−μ⁻|] (Zhang,et al., 1999 J Biomol Screening 4(2) 67-73), where μ denotes the meanand a the standard deviation. The subscript designates positive ornegative controls. The Z′ score for a robust screening assay should be≧0.50. The typical threshold=μ₊−3*σ₊. Any value that falls below thethreshold was designated a “hit”.

Dose response was analyzed using the equation:y=min+{(max−min)/(1+10^([compound]−log IC50))}, where y is the observedinitial slope, max=the slope in the absence of inhibitor, min=the slopeat infinite inhibitor, and the IC₅₀ is the [compound] that correspondsto 2 the total observed amplitude (Amplitude=max−min).

To measure modulation, activation, or inhibition of Abl KD, a testcompound was added to the assay at a range of concentrations. Inhibitorsmay inhibit Abl KD activity at an IC₅₀ in the micromolar range, thenanomolar range, or, for example, in the subnanomolar range.

Additional Kinase Assays

In addition to the c-Abl PK/LDH coupled assay (above), homogeneousluminescence-based inhibitor screening assays were developed for c-Abl,MET, AurA, and PDK1 kinases (among others). Each of these assays madeuse of an ATP depletion assay (Kinase-Glo™, Promega Corporation,Madison, Wis.) to quantitate kinase activity. The Kinase-Glo™ formatuses a thermostable luciferase to generate luminescent signal from ATPremaining in solution following the kinase reaction. The luminescentsignal is inversely correlated with the amount of kinase activity.

cAbl Luminescence-based Enzyme Assay

Materials: Abl substrate peptide=EAIYAAPFAKKK-OH (Biopeptide, San Diego,Calif.), ATP (Sigma Cat#A-3377, FW=551), HEPES buffer, pH 7.5, Bovineserum albumin (BSA) (Roche 92423420), MgCl₂, Staurosporine (Streptomycessp. Sigma Cat#85660-1 MG), white Costar 384-well flat-bottom plate (VWRCat#29444-088), Abl kinase (see below), Kinase-Glo™ (Promega Cat#V6712).

Stock Solutions: 10 mM Abl substrate peptide (13.4 mg/ml in miliQH₂O)stored at −20° C.; 100 mM HEPES buffer, pH 7.5 (5 ml 1M stock+45 mlmiliQH₂O); 10 mM ATP (5.51 mg/ml in dH₂O) stored at −20° C. (diluted 50μl into total of 10 ml miliQH₂O daily=50 μM ATP working stock); 1% BSA(1 g BSA in 100 ml 0.1 M HEPES, pH 7.5, stored at −20° C.), 100 mMMgCl₂; 200 μM Staurosporine, 2X Kinase-Glo™ reagent (made fresh orstored at −20° C.).

Standard Assay Setup for 384-well format (20 μl kinase reaction, 40 μldetection reaction): 10 mM MgCl₂; 100 μM Abl substrate peptide; 0.1%BSA; 1 μl test compound (in DMSO); 0.4 μg/ml Abl kinase domain; 10 μMATP; 100 mM HEPES buffer. Positive controls contained DMSO with no testcompound. Negative controls contained 10 μM staurosporine. The kinasereactions were initiated at time t=0 by the addition of ATP. Kinasereactions were incubated at 21° C. for 30 min, then 20 μl of Kinase-Glo™reagent were added to each well to quench the kinase reaction andinitiate the luminescence reaction. After a 20 min incubation at 21° C.,the luminescence was detected in a plate-reading luminometer.

MET Luminescence-Based Enzyme Assay

Materials: Poly Glu-Tyr (4:1) substrate (Sigma Cat# P-0275), ATP (SigmaCat#A-3377, FW=551), HEPES buffer, pH 7.5, Bovine serum albumin (BSA)(Roche 92423420), MgCl₂, Staurosporine (Streptomyces sp. SigmaCat#85660-1MG), white Costar 384-well flat-bottom plate (VWRCat#29444-088). MET kinase (see below), Kinase-Glo™ (Promega Cat#V6712).

Stock Solutions: 10 mg/ml poly Glu-Tyr in water, stored at −20° C.; 100mM HEPES buffer, pH 7.5 (5 ml IM stock+45 ml miliQH₂O); 10 mM ATP (5.51mg/ml in dH₂O) stored at −20° C. (diluted 50 μl into total of 10 mlmiliQH₂O daily=50 μM ATP working stock); 1% BSA (1 g BSA in 100 ml 0.1MHEPES, pH 7.5, stored at −20° C.), 100 mM MgCl₂; 200 μM Staurosporine,2X Kinase-Glo™ reagent (made fresh or stored at −20° C.).

Standard Assay Setup for 384-well format (20 μl kinase reaction, 40 μldetection reaction): 10 mM MgCl₂; 0.3 mg/ml poly Glu-Tyr; 0.1% BSA; 1 μltest compound (in DMSO); 0.4 μg/ml MET kinase; 10 μM ATP; 100 mM HEPESbuffer. Positive controls contained DMSO with no test compound. Negativecontrols contained 10 μM staurosporine. The kinase reactions wereinitiated at time t=0 by the addition of ATP. Kinase reactions wereincubated at 21° C. for 60 min, then 20 μl of Kinase-Glo™ reagent wereadded to each well to quench the kinase reaction and initiate theluminescence reaction. After a 20 min incubation at 21° C., theluminescence was detected in a plate-reading luminometer.

Aura Luminescence-Based Enzyme Assay

Materials: Kemptide peptide substrate=LRRASLG (Biopeptide, San Diego,Calif.), ATP (Sigma Cat#A-3377, FW=551), HEPES buffer, pH 7.5, 10% Brij35 (Calbiochem Cat#203728), MgCl₂, Staurosporine (Streptomyces sp. SigmaCat#85660-1MG), white Costar 384-well flat-bottom plate (VWRCat#29444-088), Autophosphorylated AurA kinase (see below), Kinase-Glo™(Promega Cat#V6712).

Stock Solutions: 10 mM Kemptide peptide (7.72 mg/ml in water), stored at−20° C.; 100 mM HEPES buffer+0.015% Brij 35, pH 7.5 (5 ml 1M HEPESstock+75 μL 10% Brij 35+45 ml miliQH₂O); 10 mM ATP (5.51 mg/ml in dH₂O)stored at −20° C. (diluted 50 μl into total of 10 ml miliQH₂O daily=50μM ATP working stock); 100 mM MgCl₂; 200 μM Staurosporine, 2XKinase-Glo™ reagent (made fresh or stored at −20° C.).

AurA Autophosphorylation Reaction: ATP and MgCl₂ were added to 1-5 mg/mlAurA at final concentrations of 10 mM and 100 mM, respectively. Theautophosphorylation reaction was incubated at 21° C. for 2-3 h. Thereaction was stopped by the addition of EDTA to a final concentration of50 mM, and samples were flash frozen with liquid N₂ and stored at −80°C.

Standard Assay Setup for 384-well format (20 μl kinase reaction, 40 μldetection reaction): 10 mM MgCl₂; 0.2 mM Kemptide peptide; 1 μl testcompound (in DMSO); 0.3 μg/ml Autophosphorylated AurA kinase; 10 μM ATP;100 mM HEPES+0.015% Brij buffer. Positive controls contained DMSO withno test compound. Negative controls contained 5 μM staurosporine. Thekinase reactions were initiated at time t=0 by the addition of ATP.Kinase reactions were incubated at 21° C. for 45 min, then 20 μl ofKinase-Glo™ reagent were added to each well to quench the kinasereaction and initiate the luminescence reaction. After a 20 minincubation at 21° C., the luminescence was detected in a plate-readingluminometer.

PDK1 Luminescence-Based Enzyme Assay

Materials: PDKtide peptidesubstrate=KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC (Upstate Cat# 12-401),ATP (Sigma Cat#A-3377, FW=551), HEPES buffer, pH 7.5, 10% Brij 35(Calbiochem Cat#203728), MgCl₂, Staurosporine (Streptomyces sp. SigmaCat#85660-1MG), white Costar 384-well flat-bottom plate (VWRCat#29444-088), PDK1 kinase (see below), Kinase-Glo™ (PromegaCat#V6712).

Stock Solutions: 1 mM PDKtide substrate (1 mg in 200 μl, as supplied byUpstate), stored at −20° C.; 100 mM HEPES buffer, pH 7.5 (5 ml 1M HEPESstock+45 ml miliQH₂O); 10 mM ATP (5.51 mg/ml in dH₂O) stored at −20° C.(diluted 25 μl into total of 10 ml miliQH₂O daily=25 μM ATP workingstock); 100 mM MgCl₂; 10% Brij 35 stored at 2-8° C.; 200 μMStaurosporine, 2X Kinase-Glo™ reagent (made fresh or stored at −20° C.).

Standard Assay Setup for 384-well format (20 μl kinase reaction, 40 μldetection reaction): 10 mM MgCl₂; 0.01 mM PDKtide; 1 μl test compound(in DMSO); 0.1 μg/ml PDK1 kinase; 5 μM ATP; 10 mM MgCl₂; 100 mMHEPES+0.01% Brij buffer. Positive controls contained DMSO with no testcompound. Negative controls contained 10 μM staurosporine. The kinasereactions were initiated at time t=0 by the addition of ATP. Kinasereactions were incubated at 21° C. for 40 min, then 20 μl of Kinase-Glo™reagent were added to each well to quench the kinase reaction andinitiate the luminescence reaction. After a 20 min incubation at 21° C.,the luminescence was detected in a plate-reading luminometer.

Preparation of Co-Expression Plasmid

A lambda phosphatase co-expression plasmid was constructed as follows.

An open-reading frame for Aurora kinase was amplified from a Homosapiens (human) HepG2 cDNA library (ATCC HB-8065) by the polymerasechain reaction (PCR) using the following primers:

Forward primer: TCAAAAAAGAGGCAGTGGGCTTTG Reverse primer:CTGAATTTGCTGTGATCCAGG.

The PCR product (795 base pairs expected) was gel purified as follows.The PCR product was purified by electrophoresis on a 1% agarose gel inTAE buffer and the appropriate size band was excised from the gel andeluted using a standard gel extraction kit. The eluted DNA was ligatedfor 5 minutes at room temperature with topoisomerase into pSB2-TOPO. Thevector pSB2-TOPO is a topoisomerase-activated, modified version ofpET26b (Novagen, Madison, Wis.) wherein the following sequence has beeninserted into the NdeI site: CATAATGGGCCATCATCATCATCATCACGGTGGTCATATGTCCCTT and the following sequence inserted into the BamHI site:AAGGGGGATCCTAAACTGCAGAGATCC. The sequence of the resulting plasmid, fromthe Shine-Dalgarno sequence through the “original” NdeI site, the stopsite and the “original” BamHI site is as follows:AAGGAGGAGATATACATAATGGGCCATCATCATCATCATCACGGTGGTCATATGT CCCTT [ORF]AAGGGGGATCCTAAACTGCAGAGATCC. The Aurora kinase expressed using thisvector has 14 amino acids added to the N-terminus(MetGlyHisHisHisHisHisHisGlyGlyHisMetSerLeu) and four amino acids addedto the C-terminus (GluGlyGlySer).

The phosphatase co-expression plasmid was then created by inserting thephosphatase gene from lambda bacteriophage into the above plasmid(Matsui T, et al., Biochem. Biophys. Res. Commun., 2001, 284:798-807).The phosphatase gene was amplified using PCR from template lambdabacteriophage DNA (HinDIII digest, New England Biolabs) using thefollowing oligonucleotide primers:

Forward primer (PPfor): GCAGAGATCCGAATTCGAGCTCCGTCGACGGATGGAGTGAAAGAGATGCGC Reverse primer (PPrev):GGTGGTGGTGCTCGAGTGCGGCCGCA AGCTTTCATCATGCGCCTTCTCCCTGTAC.

The PCR product (744 base pairs expected) was gel purified. The purifiedDNA and non-co-expression plasmid DNA were then digested with SacI andXhoI restriction enzymes. Both the digested plasmid and PCR product werethen gel purified and ligated together for 8 h at 16° C. with T4 DNAligase and transformed into Top10 cells using standard procedures. Thepresence of the phosphatase gene in the co-expression plasmid wasconfirmed by sequencing. For standard molecular biology protocolsfollowed here, see also, for example, the techniques described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 2001, and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley Interscience,NY, 1989.

This co-expression plasmid contains both the Aurora kinase and lambdaphosphatase genes under control of the lac promoter, each with its ownribosome binding site. By cloning the phosphatase into the middle of themultiple cloning site, downstream of the target gene, convenientrestriction sites are available for subcloning the phosphatase intoother plasmids. These sites include SacI, SalI and EcoRI between thekinase and phosphatase and HinDIII, NotI and XhoI downstream of thephosphatase.

Protein Kinase Expression

An open-reading frame for c-Abl was amplified from a Mus musculus(mouse) cDNA library prepared from freshly harvested mouse liver using acommercially available kit (Invitrogen) by PCR using the followingprimers:

Forward primer: GACAAGTGGGAAATGGAGC Reverse primer: CGCCTCGTTTCCCCAGCTC.

The PCR product (846 base pairs expected) was purified from the PCRreaction mixture using a PCR cleanup kit (Qiagen). The purified DNA wasligated for 5 minutes at room temperature with topoisomerase intopSGX3-TOPO. The vector pSGX3-TOPO is a topoisomerase-activated, modifiedversion of pET26b (Novagen, Madison, Wis.) wherein the followingsequence has been inserted into the NdeI site: CATATGTCCCTT and thefollowing sequence inserted into the BamHI site:AAGGGCATCATCACCATCACCACTGATCC. The sequence of the resulting plasmid,from the Shine-Dalgarno sequence through the stop site and the BamHI,site is as follows: AAGGAGGA GATATACATATGTC CCTT[ORF]AAGGGCATCATCACCATCACCACTGATCC. The c-Abl expressed using this vector had threeamino acids added to its N-terminus (Met Ser Leu) and 8 amino acidsadded to its C-terminus (GluGlyHisHisHisHisHisHis).

A c-Abl/phosphatase co expression plasmid was then created by subcloningthe phosphatase from the Aurora co-expression plasmid of Example 1 intothe above plasmid. Both the Aurora co-expression plasmid and the Ablnon-co-expression plasmid were digested 3 hrs with restriction enzymesEcoRI and NotI. The DNA fragments were gel purified and the phosphatasegene from the Aurora plasmid was ligated with the digested c-Abl plasmidfor 8 h at 16° C. and transformed into Top 10 cells. The presence of thephosphatase gene in the resulting construct was confirmed by restrictiondigestion analysis.

This plasmid codes for c-Abl and lambda phosphatase co expression. Ithas the additional advantage of two unique restriction sites, XbaI andNdeI, upstream of the target gene that can be used for subcloning ofother target proteins into this phosphatase co-expressing plasmid.

The plasmid for Abl T3151 was prepared by modifying the Abl plasmidusing the Quick Change mutagenesis kit (Stratagene) with themanufacturer's suggested procedure and the following oligonucleotides:

Mm05582dS4 5′-CCACCATTCTACATAATCATTGAGTTCATGAC CTATGGG-3′ Mm05582dA45′-CCCATAGGTCATGAACTCAATGATTATGTAGA ATGGTGG-3′.

Protein from the phosphatase co-expression plasmids was purified asfollows. The non-co-expression plasmid was transformed into chemicallycompetent BL21 (DE3) Codon+RIL (Stratagene) cells and the co-expressionplasmid was transformed into BL21 (DE3) pSA0145 (a strain that expressesthe lytic genes of lambda phage and lyses upon freezing and thawing(Crabtree S, Cronan J E Jr. J Bacteriol 1984 April; 158(1):354-6)) andplated onto petri dishes containing LB agar with kanamycin. Isolatedsingle colonies were grown to mid-log phase and stored at −80° C. in LBcontaining 15% glycerol. This glycerol stock was streaked on LB agarplates with kanamycin and a single colony was used to inoculate 10 mlcultures of LB with kanamycin and chloramphenicol, which was incubatedat 30° C. overnight with shaking. This culture was used to inoculate a 2L flask containing 500 ml of LB with kanamycin and chloramphenicol,which was grown to mid-log phase at 37° C. and induced by the additionof IPTG to 0.5 mM final concentration. After induction flasks wereincubated at 21° C. for 18 h with shaking.

The c-Abl T3151 KD (kinase domain) was purified as follows. Cells werecollected by centrifugation, lysed in diluted cracking buffer (50 mMTris HCl, pH 7.5, 500 mM KCl, 0.1% Tween 20, 20 mM Imidazole, withsonication, and centrifuged to remove cell debris. The soluble fractionwas purified over an IMAC column charged with nickel (Pharmacia,Uppsala, Sweden), and eluted under native conditions with a gradient of20 mM to 500 mM imidazole in 50 mM Tris, pH7.8, 50 mM NaCl, 10 mMmethionine, 10% glycerol. The protein was then further purified by gelfiltration using a Superdex 75 preparative grade column equilibrated inGF5 buffer (10 mM HEPES, pH7.5, 10 mM methionine, 500 mM NaCl, 5 mM DTT,and 10% glycerol). Fractions containing the purified c-Abl T3151 KDkinase domain were pooled. The protein obtained was 98% pure as judgedby electrophoresis on SDS polyacrylamide gels. Mass spectroscopicanalysis of the purified protein showed that it was predominantly singlyphosphorylated. The protein was then dephosphorylated with ShrimpAlkaline Phosphatase (MBI Fermentas, Burlington, Canada) under thefollowing conditions: 100 U Shrimp Alkaline Phosphatase/mg of c-AblT3151 KD, 100 mM MgCl₂, and 250 mM additional NaCl. The reaction was runovernight at 23° C. The protein was determined to be unphosphorylated byMass spectroscopic analysis. Any precipitate was spun out and thesoluble fraction was separated from reactants by gel filtration using aSuperdex 75 preparative grade column equilibrated in GF4 buffer (10 mMHEPES, pH7.5, mM methionine, 150 mM NaCl, 5 mM DTT, and 10% glycerol).

Purification of Met:

The cell pellets produced from half of a 12 L Sf9 insect cell cultureexpressing the kinase domain of human Met were resuspended in a buffercontaining 50 mM Tris-HCl pH 7.7 and 250 mM NaCl, in a volume ofapproximately 40 ml per 1 L of original culture. One tablet of RocheComplete, EDTA-free protease inhibitor cocktail (Cat# 1873580) was addedper 1 L of original culture. The suspension was stirred for 1 hour at 4°C. Debris was removed by centrifugation for 30 minutes at 39,800×g at 4°C. The supernatant was decanted into a 500 ml beaker and 10 ml of 50%slurry of Qiagen Ni-NTA Agarose (Cat# 30250) that had beenpre-equilibrated in 50 mM Tris-HCl pH 7.8, 50 mM NaCl, 10% Glycerol, 10mM Imidazole, and 10 mM Methionine, were added and stirred for 30minutes at 4° C. The sample was then poured into a drip column at 4° C.and washed with 10 column volumes of 50 mM Tris-HCl pH 7.8, 500 mM NaCl,10% Glycerol, 10 mM Imidazole, and 10 mM Methionine. The protein waseluted using a step gradient with two column volumes each of the samebuffer containing 50 mM, 200 mM, and 500 mM Imidazole, sequentially. The6× Histidine tag was cleaved overnight using 40 units of TEV protease(Invitrogen Cat# 10127017) per 1 mg of protein while dialyzing in 50 mMTris-HCl pH 7.8, 500 mM NaCl, 10% Glycerol, 10 mM Imidazole, and 10 mMMethionine at 4° C. The 6× Histidine tag was removed by passing thesample over a Pharmacia 5 ml IMAC column (Cat# 17-0409-01) charged withNickel and equilibrated in 50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10%Glycerol, 10 mM Imidazole, and 10 mM Methionine. The cleaved proteinbound to the Nickel column at a low affinity and was eluted with a stepgradient. The step gradient was run with 15% and then 80% of the B-side(A-side=50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10% Glycerol, 10 mMImidazole, and 10 mM Methionine; B-side=50 mM Tris-HCl pH 7.8, 500 mMNaCl, 10% Glycerol, 500 mM Imidazole, and 10 mM Methionine) for 4 columnvolumes each. The Met protein eluted in the first step (15%), whereasthe non-cleaved Met and the cleaved Histidine tag eluted in the 80%fractions. The 15% fractions were pooled after SDS-PAGE gel analysisconfirmed the presence of cleaved Met; further purification was done bygel filtration chromatography on an Amersham Biosciences HiLoad 16/60Superdex 200 prep grade (Cat# 17-1069-01) equilibrated in 50 mM Tris-HClpH 8.5, 150 mM NaCl, 10% Glycerol and 5 mM DTT. The cleanest fractionswere combined and concentrated to ˜10.4 mg/ml by centrifugation in anAmicon Ultra-15 10,000 Da MWCO centrifugal filter unit (Cat# UFC901024).

Purification of AurA:

The Sf9 insect cell pellets (˜18 g) produced from 6 L of cultured cellsexpressing human Aurora-2 were resuspended in 50 mM Na Phosphate pH 8.0,500 mM NaCl, 10% glycerol, 0.2% n-octyl-β-D-glucopyranoside (BOG) and 3mM β-Mercaptoethanol (BME). One tablet of Roche Complete, EDTA-freeprotease inhibitor cocktail (Cat# 1873580) and 85 units Benzonase(Novagen Cat#70746-3)) were added per 1 L of original culture. Pelletswere resuspended in approximately 50 ml per 1 L of original culture andwere then sonicated on ice with two 30-45 sec bursts (100% duty cycle).Debris was removed by centrifugation and the supernatant was passedthrough a 0.8 μm syringe filter before being loaded onto a 5 ml Ni²⁺HiTrap column (Pharmacia). The column was washed with 6 column volumesof 50 mM Na Phosphate pH 8.0, 500 mM NaCl, 10% glycerol, 3 mM BME. Theprotein was eluted using a linear gradient of the same buffer containing500 mM Imidazole. The eluant (24 ml) was cleaved overnight at 4° C. in abuffer containing 50 mM Na Phosphate pH 8.0, 500 mM NaCl, 10% glycerol,3 mM BME and 10,000 units of TEV (Invitrogen Cat# 10127-017). Theprotein was passed over a second nickel affinity column as describedabove; the flow-through was collected. The cleaved protein fractionswere combined and concentrated using spin concentrators. Furtherpurification was done by gel filtration chromatography on a S75 sizingcolumn in 50 mM Na Phosphate (pH 8.0), 250 mM NaCl, 1 mM EDTA, 0.1 mMAMP-PNP or ATP buffer, and 5 mM DTT. The cleanest fractions werecombined and concentrated to approximately 8-11 mg/ml, and were eitherflash frozen in liquid nitrogen in 120 μl aliquots and stored at −80°C., or stored at 4° C.

Purification of PDK1:

Cell pellets produced from 6 L of Sf9 insect cells expressing human PDK1were resuspended in a buffer containing 50 mM Tris-HCl pH 7.7 and 250 mMNaCl in a volume of approximately 40 mL per 1 L of original culture. Onetablet of Roche Complete, EDTA-free protease inhibitor cocktail (Cat#1873580) and 85 units Benzonase (Novagen Cat#70746-3)) were added per 1L of original culture. The suspension was stirred for 1 hour at 4° C.Debris was removed by centrifugation for 30 minutes at 39,800×g at 4° C.The supernatant was decanted into a 500 mL beaker and 10 ml of a 50%slurry of Qiagen Ni-NTA Agarose (Cat# 30250) that had beenpre-equilibrated in 50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10% Glycerol, 10mM Imidazole, and 10 mM Methionine, were added and stirred for 30minutes at 4° C. The sample was then poured into a drip column at 4° C.and washed with 10 column volumes of 50 mM Tris-HCl pH 7.8, 500 mM NaCl,10% Glycerol, 10 mM Imidazole, and 10 mM Methionine. The protein waseluted using a step gradient with two column volumes each of the samebuffer containing 50 mM, and 500 mM Imidazole, sequentially. The6×Histidine tag was cleaved overnight using 40 units of TEV protease(Invitrogen Cat# 10127017) per 1 mg of protein while dialyzing in 50 mMTris-HCl pH 7.8, 500 mM NaCl, 10% Glycerol, 10 mM Imidazole, and 10 mMMethionine at 4° C. The 6× Histidine tag was removed by passing thesample over a Pharmacia 5 ml IMAC column (Cat# 17-0409-01) charged withNickel and equilibrated in 50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10%Glycerol, 10 mM Imidazole, and 10 mM Methionine. The cleaved proteineluted in the flow-through, whereas the uncleaved protein and theHis-tag remained bound to the Ni-column. The cleaved protein fractionswere combined and concentrated using spin concentrators. Furtherpurification was done by gel filtration chromatography on an AmershamBiosciences HiLoad 16/60 Superdex 200 prep grade (Cat# 17-1069-01)equilibrated in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM DTT. Thecleanest fractions were combined and concentrated to 15 mg/ml bycentrifugation in an Amicon Ultra-15 10,000 Da MWCO centrifugal filterunit (Cat# UFC901024).

Cell Assays:

MV4-11 and THP cells were maintained in Iscove's Modified Dulbecco'sMedium supplemented with 10% fetal bovine serum (FBS) andpenicillin/streptomycin, Ba/F3 cells were maintained in RPMI 1640supplemented with 10% FBS, penicillin/streptomycin and 5 ng/mlrecombinant mouse IL-3.

Cell Survival Assays

Compounds were tested in the following assays in duplicate.

96-well XTT assay: Cells were grown in growth media containing variousconcentrations of compounds (duplicates) on a 96-well plate for 72 hoursat 37° C. The starting cell number was 5000-8000 cells per well andvolume was 120 μl. At the end of the 72-hour incubation, 40 μl of XTTlabeling mixture (50:1 solution of sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate and Electron-coupling reagent: PMS(N-methyl dibenzopyrazine methyl sulfate) were added to each well of theplate. After an additional 2-6 hours of incubation at 37° C., theabsorbance reading at 405 nm with background correction at 650 nm wasmeasured with a spectrophotometer.

384-well AlamarBlue assay: 901 of cell suspension were plated onto eachwell of a 384-well plate preprinted with 0.5 μl of compound in DMSO orDMSO only. The starting cell number was 4000 cells per well. After a72-hour incubation, 10 μl of AlamarBlue solution (440 μM resazurin inPBS) were then added to each well of the plate. After an additional2-hour incubation at 37° C., fluorescence was measured using a TECANplate reading fluorometer with excitation at 535 nm and emission at 591nm.

BCR-ABL Phospho-ELISA Assay

The following table shows the reagents that were typically used in theBCR-ABL phospho-ELISA (“P-ELISA”) assay.

TABLE 19 BCR-ABL phospho-ELISA(p-ELISA) Typical Reagent List DescriptionVendor Catalog # RPMI 1640 Invitrogen 11875-135 10% Fetal Bovine Serum,VWR 16777-014 characterized, heat inactivated Human Plasma,Bioreclamation HMPLEDTA Anticoagulant = EDTA Inc. c-Abl (Ab-3) VWR80001-286 monoclonal antibody Recombinant Mouse Chemicon IL015Interleukin-3 Adhesive Plate Seals 96well PP 325 μl round Thompson932465 bottom plate w/lid TC Instrument Co 96well Nunc Maxisorp plateFisher 12-565-136 (for colorimetric assay) Scientific 96well whiteflat-bottom Matrix 4923 plate (for luminescent assay) Lysis buffercomponents Tris-Cl pH 7.4 (20 mM) NP-40 (1%) EDTA (5 mM) Sodiumpyrophosphate (NaPP; 5 mM) NaF (5 mM) NaCl (150 mM) Protease InhibitorSigma P2714 Cocktail PMSF (1 mM) Sodium vanadate (NaVO₄; 2 mM) PBS, icecold Anti-Phosphotyrosine (4G10 ™), Upstate 16-105 or 05-321 HRPconjugate or unconjugated Goat Anti-Mouse IgG, HRP Upstate 12-349conjugate (if unconjugated 4G10 is used) BD OptEIA Reagent Set B BDBiosciences 550534 Coating Buffer (0.1M Na- carbonate, pH 9.5) AssayDiluent Wash buffer (.05% Tween/PBS) Stop Solution (2N sulfuric acid)Substrate Reagents A&B SuperSignal ELISA Pico Pierce 37070Chemiluminescent Substrate (may be used instead of Substrate ReagentsA&B)

Cells (Ba/F₃ cells transfected with WT BCR-ABL, other kinases, or T3151,Y253F, or other mutant forms of BCR-ABL) were grown in the absence ofIL-3 at least ½ week before the assay. The day before assay, the cellswere fed with fresh media so that at the time of assay the cells were inlog phase. Ba/F3 cells that had been grown in the absence of IL-3 for atleast ½ week were resuspended in RPMI 1640 so that each well of a96-well plate would contain approximately 200,000 cells. Cells weredistributed in a 96-well plate containing serially dilutedconcentrations of test compounds. Cells were typically incubated with orwithout test compounds for 60-120 minutes at 5% CO₂, 37° C. Theincubation was performed with or without other additives such as 10% FCSor 50% human plasma. After incubation of compounds, lysis buffer wasadded and incubated for 10-15 minutes; the lysate was cleared bycentrifugation.

To make the ELISA plate, commercially available Anti-ABL antibodies(e.g. (Ab-3, Calbiochem OP20) were prepared at a concentration of 0.125μg/ml in coating buffer (0.1M Na-carbonate, pH 9.5), and plated at 10 mlper plate (12.5 μl 100 μg/ml Ab/10 ml). In a high binding multi-wellplate, 100 μl Ab in coating buffer were added to each well, and eachplate was covered with a plate seal and incubated overnight at 4° C.

Excess antibody was removed and the ELISA plate was washed 3-4 timeswith 200 μl of wash buffer (0.05% Tween in PBS, pH 7.4). 150 μl oflysate (see above) were transferred to the ELISA plate. Plates weresealed and incubated 2 hours at room temperature. The detection antibody(e.g. HRP conjugated anti-pTyr or unconjugated α-p-Y 4G10, Upstate) wasprepared in assay diluent. The antibody was diluted 1:1000 (stock=2μg/μl, 200 μg in 100 μl; f.c.=2 μg/ml) in assay diluent and 10 ml ofdiluted antibody per plate were added. The lysate was removed from theELISA plates, and wells were washed four times with 200 μl of washbuffer per well. 100 μl of detection antibody was added to each well;the plate was covered, and incubated 1 hr at room temperature (21° C.).Excess detection antibody was removed from the ELISA plates, and thewells were washed four times with 200 μl of wash buffer per well.

If necessary, (i.e. for unconjugated anti-pTyr antibody) secondaryantibody (goat anti-rabbit HRP) was diluted 1:3000 in assay diluent(3.33 μl per 10 ml diluent) and added at 10 ml of diluted antibody perplate. Excess secondary antibody was removed from the ELISA plate, andthe plate was washed four times with 200 μl per well of wash buffer.

Substrate Reagent A and Substrate Reagent B (Pierce Cat#37070SuperSignal ELISA Pico Chemiluminescent Substrate) were addedimmediately before use (10 ml resultant solution per plate). 100 μlsubstrate were added per well, mixed for 1 minute, and chemiluminescentsignal was measured with a luminometer.

TABLE 20 Selected assay results: P_ELISA_[T 315I_Cells] or Ba/F3 T315IAbl_T315I_(—) Abl_Y393F proliferation MET PDK1 compound 0P IC50 IC50(XTT) AurA IC50 IC50 IC50

C C B

C C B

C C C

C C A

C C B

C C C

C C C

C C B

C C B

C C B

C C B

C C B

C C C

C C C

C C B

C C C

C C C

C C C

C C C

C C B

C C B

C C C

C C B B

C C C

C C B

C C C

C C C

C C A C

C C C B

C C C B

C C C B

C C B B

C B B

C C B B

C C AFor Table 21 above, the activity symbols represent an IC50 as follows:A>10 μM; B=1-10 μM; C<1 μM.

1. A method of inhibiting the activity of a protein kinase comprisingcontacting said protein kinase with a compound having the formula:

wherein L¹ and L² are independently a bond, —S(O)_(n)—, —O—, —NH—,unsubstituted C₁-C₅ alkylene, or unsubstituted 2 to 5 memberedheteroalkylene, wherein n is an integer from 0 to 2, and R¹ and R² areindependently substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl,or substituted or unsubstituted aryl; with the proviso that R¹ is notsubstituted or unsubstituted pyrrolyl, and that L¹ is not unsubstituted2 to 5 membered heteroalkylene when R¹ and R² are both unsubstitutedphenyl, and that L¹ is not —S(O)₂— when R² is unsubstituted piperazinyl,and that R¹ is not substituted or unsubstituted isoxazolyl when R² isunsubstituted pyridinyl.
 2. The method of claim 1, wherein said proteinkinase is an Abelson tyrosine kinase, Ron receptor tyrosine kinase, Metreceptor tyrosine kinase, Fms-like tyrosine kinase-3, Aurora kinases,p21-activated kinase-4, or 3-phosphoinositide-dependent kinase-1.
 3. Themethod of claim 1, wherein said protein kinase is a Bcr-Abl kinasehaving a mutation selected from the group consisting of M244V, L248V,G250E, G250A, Q252H, Q252R, Y253F, Y253H, E255K, E255V, D276G, F311L,T315I, T315N, T315A, F317V, F317L, M343T, M351T, E355G, F359A, F359V,V3791, F382L, L387M, H396P, H396R, S417Y, E459K and F486S.
 4. The methodof claim 3, wherein said protein kinase has a T315I mutation.
 5. Apharmaceutical composition comprising one or more pharmaceuticallyacceptable excipients and a compound having the formula:

wherein L¹ and L² are independently a bond, —S(O)_(n)—, —O—, —NH—,unsubstituted C₁-C₅ alkylene, or unsubstituted 2 to 5 memberedheteroalkylene, wherein n is an integer from 0 to 2, and R¹ and R² areindependently substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl,or substituted or unsubstituted aryl; with the proviso that R¹ is notsubstituted or unsubstituted pyrrolyl, and that L¹ is not unsubstituted2 to 5 membered heteroalkylene when R¹ and R² are both unsubstitutedphenyl, and that L¹ is not —S(O)₂— when R² is unsubstituted piperazinyl,and that R¹ is not substituted or unsubstituted isoxazolyl when R² isunsubstituted pyridinyl.
 6. The method of claim 1, wherein L¹ and L² areindependently a bond, —S(O)_(n)—, —O—, —NH—, or unsubstituted C₁-C₅alkylene.
 7. The method of claim 1, wherein L¹ and L² are a bond.
 8. Themethod of claim 1, wherein L¹ or L² is a bond.
 9. The method of claim 1,wherein R¹ is substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted 5 or 6membered heteroaryl, or substituted or unsubstituted aryl.
 10. Themethod of claim 1, wherein R¹ is substituted or unsubstituted 6 memberedheteroaryl, or substituted or unsubstituted aryl.
 11. The method ofclaim 1, wherein R¹ is (1) unsubstituted C₃-C₇ cycloalkyl; (2)unsubstituted 3 to 7 membered heterocycloalkyl; (3) unsubstitutedheteroaryl; (4) unsubstituted aryl; (5) substituted C₃-C₇ cycloalkyl;(6) substituted 3 to 7 membered heterocycloalkyl; (7) substituted aryl;or (8) substituted heteroaryl; wherein (5) and (6) are substituted withan oxo, —OH, —CF₃, —COOH, cyano, halogen, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, R¹²-substituted or unsubstitutedheteroaryl, -L¹²-C(X¹)R⁷, -L¹²-OR⁸, -L¹²-NR⁹¹R⁹², or -L¹²-S(O)_(m)R¹⁰,(7) and (8) are substituted with an —OH, —CF₃, —COOH, cyano, halogen,R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R₁₁-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl,R¹²-substituted or unsubstituted heteroaryl, -L¹²-C(X¹)R⁷, -L¹²-OR⁸,-L¹²-NR⁹¹R⁹², or L¹²S(O)_(m)R¹⁰, wherein (a) X¹ is ═S, ═O, or ═NR¹⁵,wherein R¹⁵ is H, —OR¹⁵¹, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, or R¹²-substituted or unsubstituted heteroaryl,wherein R¹⁵¹ is hydrogen or R¹¹-substituted or unsubstituted C₁-C₁₀alkyl, (b) m is an integer from 0 to 2; (c) R⁷ is hydrogen,R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl,R¹²-substituted or unsubstituted heteroaryl, —OR⁷¹, or —NR⁷²R⁷³, whereinR⁷¹, R⁷², and R⁷³ are independently hydrogen, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, or R¹²-substituted orunsubstituted heteroaryl, wherein R⁷² and R⁷³ are optionally joined withthe nitrogen to which they are attached to form an R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R¹²-substituted orunsubstituted heteroaryl; (d) R⁸, R⁹¹ and R⁹² are independentlyhydrogen, —CF₃, R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl,R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl,—C(X²)R⁸¹, or —S(O)_(w)R⁸¹, wherein R⁹¹ and R⁹² are optionally joinedwith the nitrogen to which they are attached to form an R¹¹-substitutedor unsubstituted 3 to 7 membered heterocycloalkyl, or R¹²-substituted orunsubstituted heteroaryl, wherein (i) X² is ═S, ═O, or ═NR¹⁶, whereinR¹⁶ is R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl, orR¹²-substituted or unsubstituted heteroaryl; (ii) w is an integer from 0to 2, and (iii) R⁸¹ is hydrogen, R¹¹-substituted or unsubstituted C₁-C₁₀alkyl, R¹¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹²-substituted orunsubstituted aryl, R¹²-substituted or unsubstituted heteroaryl, or—NR⁸¹¹R⁸¹², wherein R⁸¹¹ and R⁸¹² are independently hydrogen,R1¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl, orR¹²-substituted or unsubstituted heteroaryl, wherein R⁸¹¹ and R⁸¹² areoptionally joined with the nitrogen to which they are attached to forman R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR¹²-substituted or unsubstituted heteroaryl; (e) R¹⁰ is hydrogen,R¹¹-substituted or unsubstituted C₁-C₁₀ alkyl, R¹¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R¹¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R¹¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R¹²-substituted or unsubstituted aryl,R¹²-substituted or unsubstituted heteroaryl, or —NR¹⁰¹R¹⁰², wherein (i)R¹⁰¹ and R¹⁰² are independently hydrogen, R¹¹-substituted orunsubstituted C₁-C₁₀ alkyl, R¹¹-substituted or unsubstituted 2 to 10membered heteroalkyl, R¹¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹²-substituted or unsubstituted aryl, or R¹²-substituted orunsubstituted heteroaryl, wherein R¹⁰¹ and R¹⁰² are optionally joinedwith the nitrogen to which they are attached to form an R¹¹-substitutedor unsubstituted 3 to 7 membered heterocycloalkyl, or R¹²-substituted orunsubstituted heteroaryl; (f) L¹² is a bond, unsubstituted C₁-C₁₀alkylene, or unsubstituted heteroalkylene; (g) R¹¹ is oxo, —OH, —COOH,—CF₃, —OCF₃, —CN, amino, halogen, R¹³-substituted or unsubstituted 2 to10 membered alkyl, R¹³-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R¹³-substituted or unsubstituted C₃-C₇ cycloalkyl,R¹³-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R¹⁴-substituted or unsubstituted aryl, or R¹⁴-substituted orunsubstituted heteroaryl; (h) R¹² is —OH, —COOH, amino, halogen, —CF₃,—OCF₃, —CN, R¹³-substituted or unsubstituted 2 to 10 membered alkyl,R¹³-substituted or unsubstituted 2 to 10 membered heteroalkyl,R¹³-substituted or unsubstituted C₃-C₇ cycloalkyl, R¹³-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R¹⁴-substituted orunsubstituted aryl, or R¹⁴-substituted or unsubstituted heteroaryl; (i)R¹³ is oxo, —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇ cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl; and (j) R¹⁴ is —OH, —COOH,amino, halogen, —CF₃, —OCF₃, —CN, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.
 12. The method of claim 1,wherein R¹ is substituted or unsubstituted 6 membered heteroaryl, orsubstituted or unsubstituted aryl.
 13. The method of claim 1, wherein L¹is a bond.
 14. The method of claim 11, wherein R¹ is (7) or (8), wherein(7) and (8) are substituted with an —OH, —CF₃, halogen, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstitutedC₃-C₇, cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, or -L¹²-OR⁸, wherein L¹²is a bond.
 15. The method of claim 14, wherein R⁸ is CF₃.
 16. The methodof claim 11, wherein R¹ is (7) or (8), wherein (7) and (8) aresubstituted with an —OCH₃, —OCF₃, —CH₃, —CF₃, —OCH₂CH₃, halogen, orcyclopropyloxy.
 17. The method of claim 11 wherein L¹ and L² are a bond.18. The method of claim 1, wherein R² is (1) unsubstituted C₃-C₇cycloalkyl; (2) unsubstituted 3 to 7 membered heterocycloalkyl; (3)unsubstituted heteroaryl; (4) unsubstituted aryl; (5) substituted C₃-C₇cycloalkyl; (6) substituted 3 to 7 membered heterocycloalkyl; (7)substituted aryl; or (8) substituted heteroaryl; wherein (5) and (6) aresubstituted with an oxo, —OH, —CF₃, —COOH, cyano, halogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, -L²²-C(X³)R³, -L²²-OR⁴,-L²²-NR⁵¹R⁵², or -L²²S(O)_(q)R⁶, (7) and (8) are substituted with an—OH, —CF₃, —COOH, cyano, halogen, R²¹-substituted or unsubstitutedC₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, R²²-substituted or unsubstitutedheteroaryl, -L²²-C(X³)R³, -L²²OR⁴, -L²²-NR⁵¹R⁵², or -L²²S(O)_(q)R⁶,wherein (a) X₃ is S, ═O, or ═NR¹⁷, wherein R¹⁷ is H, —OR¹⁷¹,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, wherein R¹⁷¹ is H orR²¹-substituted or unsubstituted C₁-C₁₀ alkyl; (b) q is an integer from0 to 2; (c) R³ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀alkyl, R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl, —OR³¹,or —NR³²R³³, wherein (i) R³¹, R³², and R³³ are independently hydrogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, wherein R³² and R³³ areoptionally joined with the nitrogen to which they are attached to forman R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR²²-substituted or unsubstituted heteroaryl; (d) R⁴, R⁵¹ and R⁵² areindependently hydrogen, —CF₃, R²¹-substituted or unsubstituted C₁-C₁₀alkyl, R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl,—C(X⁴)R⁴¹, or —S(O)_(v)R⁴¹, wherein R⁵¹ and R⁵² are optionally joinedwith the nitrogen to which they are attached to form an R²¹-substitutedor unsubstituted 3 to 7 membered heterocycloalkyl, or R²²-substituted orunsubstituted heteroaryl, wherein (ii) X⁴ is ═S, ═O, or ═NR¹⁸, whereinR¹⁸ is R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl; (ii) v is an integer from 0to 2; (iii) R⁴¹ is hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀alkyl, R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, R²²-substituted or unsubstituted heteroaryl, or—NR⁴¹¹R⁴¹², wherein R⁴¹¹ and R⁴¹² are independently selected fromhydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substitutedor unsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇ cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, wherein R⁴¹¹ and R⁴¹² areoptionally joined with the nitrogen to which they are attached to forman R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR²²-substituted or unsubstituted heteroaryl; (e) R⁶ is hydrogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇, cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl,R²²-substituted or unsubstituted heteroaryl, or —NR⁶¹R⁶², wherein (i)R⁶¹ and R⁶² are hydrogen, R²¹-substituted or unsubstituted C₁-C₁₀ alkyl,R²¹-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl, R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²²-substituted orunsubstituted aryl, or R²²-substituted or unsubstituted heteroaryl,wherein R⁶¹ and R⁶² are optionally joined with the nitrogen to whichthey are attached to form an R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, or R²²-substituted or unsubstitutedheteroaryl; (f) L²² is a bond, unsubstituted C₁-C₁₀ alkylene orunsubstituted heteroalkylene; (g) R²¹ is oxo, —OH, —COOH, —CF₃, —OCF₃,—CN, amino, halogen, R²³-substituted or unsubstituted 2 to 10 memberedalkyl, R²³-substituted or unsubstituted 2 to 10 membered heteroalkyl,R²³-substituted or unsubstituted C₃-C₇ cycloalkyl, R²³-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, R²⁴-substituted orunsubstituted aryl, or R²⁴-substituted or unsubstituted heteroaryl; (h)R²² is —OH, —COOH, amino, halogen, —CF₃, —OCF₃, —CN, R²³-substituted orunsubstituted 2 to 10 membered alkyl, R²³-substituted or unsubstituted 2to 10 membered heteroalkyl, R²³-substituted or unsubstituted C₃-C₇cycloalkyl, R²³-substituted or unsubstituted 3 to 7 memberedheterocycloalkyl, R²⁴-substituted or unsubstituted aryl, orR²⁴-substituted or unsubstituted heteroaryl; (i) R²³ is oxo, —OH, —COOH,amino, halogen, —CF₃, —OCF₃, —CN, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl; and (j) R²⁴ is —COOH,amino, halogen, —CF₃, —OCF₃, —CN, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.
 19. The method of claim18, wherein L² is a bond.
 20. The method of claim 18, wherein R² is (3),(4), (7), or (8).
 21. The method of claim 19, wherein R² is (7) or (8).22. The method of claim 21, wherein (7) and (8) are substituted with an-L²²-C(X³)R³, -L²²-OR⁴, -L²²-NR⁵¹R⁵², -L²²C(NH)NR³²R³³, or-L²²-S(O)_(q)R⁶.
 23. The method of claim 22, wherein R³ is —NR³²R³³; X³is ═O or ═NR¹⁷; R⁶ is —NR⁶¹R⁶²; R⁵¹ is —C(O)R⁴¹ or —S(O)_(v)R⁴¹.
 24. Themethod of claim 23, wherein R⁴¹ is —NR⁴¹¹R⁴¹².
 25. The method of claim19, wherein R² is (7) or (8), wherein (7) and (8) are substituted with—OH, —CF₃, —COOH, amino, halogen, unsubstituted 2 to 10 memberedheteroalkyl, unsubstituted C₃-C₇ cycloalkyl, unsubstituted 3 to 7membered heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,or -L²²-C(X³)R³, wherein X³ is ═O; R³ is unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₇cycloalkyl, unsubstituted 3 to 7 membered heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl, or —NR³²R³³, wherein R³²and R³³ are independently hydrogen, R²¹-substituted or unsubstitutedC₁-C₁₀ alkyl, R²¹-substituted or unsubstituted 2 to 10 memberedheteroalkyl, R²¹-substituted or unsubstituted C₃-C₇ cycloalkyl,R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl,R²²-substituted or unsubstituted aryl, or R²²-substituted orunsubstituted heteroaryl, wherein R³² and R³³ are optionally joined withthe nitrogen to which they are attached to form an R²¹-substituted orunsubstituted 3 to 7 membered heterocycloalkyl, or R²²-substituted orunsubstituted heteroaryl.
 26. The method of claim 19, wherein R² is (7)or (8), wherein (7) and (8) are substituted with unsubstituted 2 to 10membered heteroalkyl, or -L²²-C(O)R³, wherein L²² is a bond; and R³ is—NR³²R³³, wherein R³² and R³³ are independently hydrogen,R²¹-substituted or unsubstituted C₁-C₁₀ alkyl, R²¹-substituted orunsubstituted 2 to 10 membered heteroalkyl, R²¹-substituted orunsubstituted C₃-C₇, cycloalkyl, R²¹-substituted or unsubstituted 3 to 7membered heterocycloalkyl, R²²-substituted or unsubstituted aryl, orR²²-substituted or unsubstituted heteroaryl, wherein R³² and R³³ areoptionally joined with the nitrogen to which they are attached to forman R²¹-substituted or unsubstituted 3 to 7 membered heterocycloalkyl, orR²²-substituted or unsubstituted heteroaryl.
 27. The method of claim 1,wherein R¹ is a substituted or unsubstituted fused ring aryl orsubstituted or unsubstituted fused ring heteroaryl.
 28. The method ofclaim 1, wherein R² is substituted or unsubstituted indolyl, substitutedor unsubstituted quinolinyl, or substituted or unsubstitutedbenzodioxolyl.
 29. The method of claim 1, wherein R² is a substituted orunsubstituted fused ring aryl or substituted or unsubstituted fused ringheteroaryl.
 30. The method of claim 1, wherein R² is substituted orunsubstituted indolyl, substituted or unsubstituted quinolinyl, orsubstituted or unsubstituted benzodioxolyl.
 31. The method of claim 1,wherein R¹ and R² are independently substituted or unsubstitutedhydantoinyl, substituted or unsubstituted dioxolanyl, substituted orunsubstituted dioxanyl, substituted or unsubstituted trioxanyl,substituted or unsubstituted tetrahydrothienyl, substituted orunsubstituted tetrahydrofuranyl, substituted or unsubstitutedtetrahydrothiophenyl, substituted or unsubstituted tetrahydropyranyl,substituted or unsubstituted tetrahydrothiopyranyl, substituted orunsubstituted pyrrolidinyl, substituted or unsubstituted morpholino,substituted or unsubstituted piperidinyl, substituted or unsubstitutedpyrazolyl, substituted or unsubstituted furanyl, substituted orunsubstituted imidazolyl, substituted or unsubstituted isoxazolyl,substituted or unsubstituted oxadiazolyl, substituted or unsubstitutedoxazolyl, substituted or unsubstituted pyridyl, substituted orunsubstituted pyrazyl, substituted or unsubstituted pyrimidyl,substituted or unsubstituted pyridazinyl, substituted or unsubstitutedthiazolyl, substituted or unsubstituted isothioazolyl, substituted orunsubstituted triazolyl, substituted or unsubstituted thienyl,substituted or unsubstituted triazinyl, substituted or unsubstitutedthiadiazolyl, or substituted or unsubstituted tetrazolyl.
 32. The methodof claim 7, wherein R¹ and R² are independently substituted orunsubstituted aryl.
 33. The method of claim 32, wherein R¹ and R² areindependently substituted or unsubstituted phenyl.
 34. The method ofclaim 33, wherein R¹ and R² are each substituted phenyl.
 35. The methodof claim 34, wherein the substituents are selected from halogen, C₁-C₃unsubstituted alkyl, —C(═O)NR⁷²R⁷³, —OR⁸ and —NR⁹¹R⁹².